Genetically modified reduced-browning fruit-producing plant and produced fruit thereof, and method of obtaining such

ABSTRACT

A genetically modified fruit-producing plant, said plant having sufficiently reduced total Polyphenol Oxidase (PPO) activity relative to a wild type of said plant to reduce browning in the fruit of said plant relative to said wild type, wherein the reduced total PPO activity results from a reduction in activity of at least two PPO isoenzymes in said plant relative to said wild type, or a cell, seed, seedling, part, tissue, cell, fruit or progeny of said plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/404,236, filed Jan. 12, 2017, which is a continuation of U.S.application Ser. No. 14/049,952, filed Oct. 9, 2013, which is acontinuation of U.S. application Ser. No. 12/919,735, filed Aug. 26,2010, which is the National Stage of International Application No.PCT/CA2009/000212, filed Feb. 26, 2009, which claims the benefit ofProvisional Application No. 61/031,821, filed Feb. 27, 2008, thedisclosures of which are expressly incorporated herein by reference intheir entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 53005-3C3_SEQ_2019-01-15.txt. The file is 103KB; was created on Jan. 15, 2019; and is being submitted via EFS-Webwith the filing of the specification.

FIELD OF THE INVENTION

This invention relates to a genetically modified fruit-producing plant,plant cell, seed, seedling, progeny thereof, or produced fruit thereof,which plant produces a reduced-browning fruit. This invention furtherrelates to a method of obtaining such.

BACKGROUND OF THE INVENTION

Browning of Fruit

Browning of apples and other fruit from damage, such as cuts, bruises,slicing, juicing, cell death, or any other form of damage that disruptscell membranes, is believed to be caused by the enzymatic reactioncatalyzed by Polyphenol Oxidase (PPO). The brown pigment is a polymerformed from the non-enzymatic condensation of quinones, with lesseramounts of amino acids and proteins, into lignin-like compounds. Thequinones are synthesized from di-phenols in the enzymatic reactioncatalyzed by PPO (Whitaker and Lee, 1995). Most PPOs also havemonophenolase activity and convert monophenols to di-phenols (Mar-Sojoet al., 1998). The cause of browning has been understood for some time,yet the solution to reducing browning remains an on-going problem forindustry.

Browning reduces quality by causing detrimental flavor and nutritionalchanges (Elkin, 1990). With the explosive growth of the fresh-cutproduce sector, lost opportunities have become more evident given that,for instance, browning limits the use of apple and other fruits incommercial fresh cut produce products. It is thus a significant problemlimiting the widespread introduction of fresh cut produce products suchas prepared apple slices.

Browning is also a major consideration in the manufacture of juice, andbrown bruises are a significant cause of reduced grade for growers andof lost value for institutional processors (restaurants, hospitals,etc.) and retailers, who have to accept these losses or try to minimizethem through the implementation of improved handling practices.

Approaches to Control Browning

Various approaches to control vegetable and fruit browning have beendescribed and resulted in mixed success, due to a variety of reasons,including cost and amount of handling. For a general review ofstrategies for reducing fruit browning, see e.g.: Friedman (1991),Iyengar and McEvily, (1992), Whitaker and Lee (1995), McEvily et al.(1992), Sapers (1993), Weemaes (1998), Martinez and Whitaker (1995), andBrushett (2006).

U.S. Pat. No. 5,939,117 (Cheng et al.) and U.S. Pat. No. 5,925,395(Cheng) describes an anti-browning/anti-oxidant dip treatment. Fresh-cutapple slices which have been treated with an anti-browning/anti-oxidantdip, are described as having reduced browning. However, theoff-flavoring and high cost of the anti-browning/anti-oxidant dipsolution has limited their commercial success. Furthermore, anti-oxidantdip solutions do not deal effectively with secondary browning thatresults from the cutting knife and skin deformation prior to cutting andother secondary browning reactions, which lead to a thin brown lineunder the skin on the apple slice and other market detractingattributes.

Other approaches to control browning have been described, including butnot limited to, cultivation in low oxygen atmosphere and low temperature(Heimdal et al., 1995); treatment with calcium ascorbate, glutathione,cysteine and citrate (Jiang et al., 1998); treatment with sulfites andsub optimal pH and high-pressure carbon dioxide (Chen et al., 1992);treatment of fresh cut apple slices with natural products (Ruta et al.,1999); and a treatment with a 10% solution of honey (Osmianski and Lee,1990).

Murata et al. (2000 and 2001) report that by suppression of a single PPOgene homologous to the apple PPO gene APOS they obtained apple shootsand callus having reduced PPO activity which exhibit low browningpotential in vitro. However, these references do not disclose a reducedbrowning fruit-producing plant or reduced-browning apple nor whethersuppression of a single PPO gene homologous to APOS would be sufficientto obtain such a reduced-browning fruit-producing plant orreduced-browning apple.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a genetically modifiedfruit-producing plant, said plant having sufficiently reduced totalPolyphenol Oxidase (PPO) activity relative to a wild type of said plantto reduce browning in the fruit of said plant relative to said wildtype, wherein the reduced total PPO activity results from a reduction inactivity of at least two PPO isoenzymes in said plant relative to saidwild type, or a cell, seed, seedling, part, tissue, cell, fruit orprogeny of said plant.

In another aspect, the invention relates to a method for producing agenetically modified fruit-producing plant, said plant havingsufficiently reduced total Polyphenol Oxidase (PPO) activity relative toa wild type of said plant to reduce browning in the fruit of said plantrelative to said wild type, said method comprising reducing the activityof at least two PPO isoenzymes in said plant relative to said wild type.

In another aspect, the invention relates to a nucleic acid constructcomprising: a promoter; a first nucleic acid sequence comprising atleast 200 contiguous nucleotides of a nucleic acid molecule encoding apolypeptide of SEQ ID NO: 19; a second nucleic acid sequence comprisingat least 200 contiguous nucleotides of a nucleic acid molecule encodinga polypeptide of SEQ ID NO: 21; a third nucleic acid sequence comprisingat least 200 contiguous nucleotides of a nucleic acid molecule encodinga polypeptide of SEQ ID NO: 23; and a fourth nucleic acid sequencecomprising at least 200 contiguous nucleotides of a nucleic acidmolecule set forth in SEQ ID NO: 24 and encoding a polypeptide of SEQ IDNO: 25; wherein the first, second, third and fourth nucleic acidmolecules are operably linked to said promoter in sense orientation.

In another aspect, the invention relates to a nucleic acid constructencoding an mRNA capable of forming a stem loop structure, the nucleicacid construct comprising, from 5′ to 3′: a promoter, a first set ofnucleic acid sequences, a spacer, and a second set of nucleic acidsequences, said first set of nucleic acid sequences comprising: a firstnucleic acid sequence comprising at least 200 contiguous nucleotides ofa nucleic acid molecule encoding a polypeptide of SEQ ID NO: 19; asecond nucleic acid sequence comprising at least 200 contiguousnucleotides of a nucleic acid molecule encoding a polypeptide of SEQ IDNO: 21; a third nucleic acid sequence comprising at least 200 contiguousnucleotides of a nucleic acid molecule encoding a polypeptide of SEQ IDNO: 23; and a fourth nucleic acid sequence comprising at least 200contiguous nucleotides of a nucleic acid molecule encoding a polypeptideof SEQ ID NO: 25; wherein the first, second, third and fourth nucleicacid sequences are operably linked to said promoter in senseorientation; said second set of nucleic acid sequences comprising: saidfirst, second, third and fourth nucleic acid sequences operably linkedto said promoter in anti-sense orientation; wherein, the first andsecond sets of nucleic acid molecules are separated by the spacer.

In another aspect, the invention relates to a genetically modified plantcell transformed with the nucleic acid construct as described above.

In another aspect, the invention relates to a genetically modified plantcomprising the genetically modified plant cell as described above.

Other aspects and features of the present invention will become apparentto those of ordinary skill in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, which illustrate, by way of example only, embodiments ofthe present invention:

FIG. 1A-1D show nucleic acid sequence and amino acid sequence alignmentsbetween AP14 [SEQ ID NO:2 and 4] and PGO3 [SEQ ID NO:1 and 3], withClustal W™ (1.83).

FIG. 2A-2E show cloned novel partial sequences and/or nucleic acidsequence fragments of known apple PPO isoenzyme encoding sequences: APO5[SEQ ID NO: 5]; PPO3 [SEQ ID NO:6]; PPO7 [SEQ ID NO: 7]; PPO2 [SEQ IDNO: 8]; PPOJ [SEQ ID NO:9]; GPO3 [SEQ ID NO:10]; AP14 [SEQ ID NO:11];pSR7 [SEQ ID NO:12]; APO3 5′ [SEQ ID NO:13]; APO9 5′ [SEQ ID NO:14];APO3 3′ [SEQ ID NO:15]; APO9 3′ [SEQ ID NO:16]; and pSR8 [SEQ ID NO:17].

FIG. 3 shows sequence identity (%) obtained through a multiple nucleicacid sequence alignments of apple PPO encoding sequences with Clustal W™(1.82).

FIG. 4A-4D show nucleic acid sequence and amino acid sequence of fourapple PPO encoding sequences: PPO2 [SEQ ID NO: 18] PPO2 (translation)[SEQ ID NO:19]; GPO3 [SEQ ID NO:20]; GPO3 (translation) [SEQ ID NO:21];APO5 [SEQ ID NO:22]; APO5 (translation) [SEQ ID NO:23]; pSR7 [SEQ IDNO:24]; and pSR7 (translation) [SEQ ID NO:25].

FIG. 5A-5B: A. shows nucleic acid fragment of four PPO encodingsequences used for constructing PGAS: PPO2 [SEQ ID NO: 26]; GPO3 [SEQ IDNO:27]; APO5 [SEQ ID NO: 28]; and PSR7 [SEQ ID NO:29]. B. shows thenucleic acid fragments used in the construction of PGAS2: PPO2 [SEQ IDNO:30]; GPO3 [SEQ ID NO:31]; APO5 [SEQ ID NO:32]; PSR7 [SEQ ID NO: 33];and ACO2 [SEQ ID NO:34].

FIG. 6A-6B. A. shows the nucleic acid sequence of the PGAS transgenefragment in the sense orientation [SEQ ID NO:35]. B. shows the nucleicacid sequence of the PGAS2 transgene fragment [SEQ ID NO:36].

FIG. 7A-7X. A-L show nucleic acid sequence alignment between the applePPO isoenzyme encoding genomic sequences and the corresponding nucleicacid sequences used for constructing PGAS: PPO2 [SEQ ID NO:37] and PPO2PGAS [SEQ ID NO:38]; GPO3 [SEQ ID NO: 39] and GPO3 PGAS [SEQ ID NO:40];APO5 [SEQ ID NO:41] and APO5_PGAS [SEQ ID NO:42]; pSR7 [SEQ ID NO:43]and pSR7 PGAS [SEQ ID NO:44]. M-X show nucleic acid sequence alignmentbetween the apple PPO isoenzyme encoding genomic sequences and thecorresponding nucleic acid sequences used for constructing PGAS2: PPO2[SEQ ID NO:45] and PPO2 PGAS2 [SEQ ID NO:46]; GPO3 [SEQ ID NO:47] andGPO3 PGAS2 [SEQ ID NO:48]; APOS [SEQ ID NO:49] and APO5_PGAS2 [SEQ IDNO:50]; pSR7 [SEQ ID NO:51] and pSR7_PGAS2 [SEQ ID NO:52].

FIG. 8A-8B: A. shows an illustrative representation of GEN-03 comprisingthe PGAS transgene fragment in the sense orientation; and B. illustratesOSF-02, where P=PPO2; G=GPO3; A=APO5; and S=pSR7.

FIG. 9A-9E: A-C show nucleic acid sequence of the T-DNA elements ofGEN-03 which are typically transferred into a plant following atransformation event: LB [SEQ ID NO:53]; P_(NOS) [SEQ ID NO:54]; nptII[SEQ ID NO:55]; T_(NOS) [SEQ ID NO:56]; P_(CAMV35S) [SEQ ID NO:57]; PGAS[SEQ ID NO: 58]; T_(NOS) [SEQ ID NO:59]; and RB [SEQ ID NO:60]. D-Eshows the nucleic acid sequence of the elements for PGAS2: RB [SEQ IDNO:61]; P_(BUL409s) [SEQ ID NO:62]; PGAS2 [SEQ ID NO:63]; T_(UBI3) [SEQID NO:64]; and LB [SEQ ID NO: 65].

FIG. 10A-10D show PPO suppression or activity in examples of GEN-03 (Aand B) and OSF-02 (C and D) genetic events.

FIG. 11 shows an illustrative example of the results obtained from adetailed examination of the reduced-browning phenotype of genetic eventssent to field trial.

FIG. 12 shows an illustrative example of a controlled bruised responseof genetic events (743 and 784) sent to field trial relative to controlevents.

FIG. 13 shows an illustrative example of a reduced-browning phenotype ofjuice produced from genetic events (743 and 784) sent to field trialrelative to control events.

FIG. 14A-14C show an illustrative example of measurement of PPO geneexpression, total PPO activity, and change in luminosity in immaturefruit obtained from genetic events sent to field trial, in twoindependent experiments (I and II).

FIG. 15 shows the nucleic acid sequence of three pear PPO encodingsequences: PearGB [SEQ ID NO:66]; Pearl [SEQ ID NO:67] and Pear2 [SEQ IDNO:68].

FIG. 16 shows sequence identity (%) obtained through a multiple nucleicacid sequence alignment of apple and pear PPO encoding sequences withClustal W™ (1.82).

FIG. 17 shows the relationship between PPO activity in tissue cultureleaf material (TC), PPO activity in immature fruit tissue (2005), ImpactBruising (2005 and 2006) and PPO gene expression in immature fruit(2005).

DETAILED DESCRIPTION

The invention provides a genetically modified fruit-producing plant,seed, seedling, progeny thereof, or produced fruit thereof, whichgenetically modified plant produces a reduced-browning fruit by having areduced Polyphenol Oxidase (PPO) activity relative to controlfruit-producing plant, seed, seedling, progeny thereof, or producedfruit thereof. The invention also provides a method of obtaining suchgenetically modified fruit-producing plant.

Polyphenol Oxidase (PPO) Expression in Plants

PPOs are copper-containing metalloenzymes that catalyze the oxidation ofphenols to produce quinones. Quinones may subsequently react with aminoacids and proteins to form brown and black pigments, resulting in thebrowning of produce, including without limitation, fruits andvegetables. PPOs are localized to the plastid in plants, whereas thephenolic substrates of the enzymes are sequestered in vacuoles. Thiscompartmentalization prevents a PPO from reacting with its substrateunless the plant cells are damaged and the enzyme and its substrate aremixed.

PPO gene expression has been described in diverse genera of animals,plants, fungi and bacteria (for a review, see Mayer (2006)).

Any plant that expresses PPO and is susceptible to bruising, or producesfruit that is susceptible to bruising may be used in the context of theinvention.

Among plants, PPO has been described, without limitation, infruit-producing plants (e.g. apricot, apple, banana, pear), vegetables(e.g. artichoke, cabbage, potato, tomato), flowers (e.g. orchid); herbs(e.g. oregano); grains (e.g. wheat); and others (e.g. red clover).

The invention has application in any fruit-producing plant thatexpresses PPO. Such plants include, without limitation, apple, apricot,avocado, banana, blackberry, blueberry, cherry, cranberry, custardapple, date, durian, fig, grapefruit, grape, jack fruit, kiwi fruit,lychee, mandarin, mangosteen, mango, melon, nashi, nectarine, orange,papaya or paw paw, passionfruit, peach, pear, persimmon, pineapple,plum, pomegranate, pomelo, raspberry, rhubarb, star fruit, strawberry,tamarillo, and tangerine plants or trees (as used herein the term“plant” is intended to encompass also fruit-producing trees). Theinvention encompasses also cells, seeds, seedlings, parts, tissues,fruit and progeny of such plants.

In one embodiment the plant is an apple plant. The following areexamples of PPO genes described in apple, and which may be targeted forreduction of PPO activity:

U.S. Pat. No. 6,936,748 (Robinson and Dry) described the cloning of PPOgenes from potato tubers, grape, apple and broad bean. Apple PPO genespSR7 and pSR8 were identified in addition to a partial sequence of agenomic clone, GALPO3, which appears to be very similar to the apple PPOgene, APO3.

Boss et al. (1995) screened an apple cDNA library with pSR8 and isolatedsix clones, including the PPO gene APO5. In personal communications withBoss, it was noted that other apple PPO clones, APO1, APO2, APO3 andAPO9, were similar or identical to each other and 70% identical to APO5.

Haruta et al. (1998) isolated and characterized two apple PPO clonesPPO3 and PPO7 that are nearly identical to APOS.

Kim et al. (2001) teaches that apple PPO gene PPO2 is 96% identical topSR8. PPO2 has less homology with the other apple isoenzymes than theyhave to each other. PPO2 is not closely related to peach or cherry PPOsbut somewhat related to a PPO sequence from apricot.

In many plant species, PPO genes are organized in multigene families.For example, Kruger et al (1976) reported 12 isoenzymes of PPO in wheat.Newman et al (1993) reported at least six PPO genes in tomato, withhomologies ranging from 70-96%. Boss et al (1995) reported at least fourPPO genes in apple.

The invention involves reducing activity of at least two PPO isoenzymesin a fruit-producing plant. As used herein, the term “PPO isoenzyme”encompasses enzymes that differ in amino acid sequence but catalyze thesame chemical reaction. In biochemistry, isoenzymes (or isozymes) areisoforms (closely related variants) of enzymes. In many cases,homologous genes encode isoenzymes.

In an embodiment, the plant is an apple plant (Malus x domestica). Theinvention may be practiced in any variety of apple, such as, forexample, Golden Delicious, Granny Smith, Fuji, Gala, MacIntosh, PPOisoenzymes in apple, the activity of which may be reduced in accordancewith the invention, include, without limitation, any two or more of APO5(Boss et al. (1995); pSR7; (Robinson (1993)); pSR8 (PPO2) (Robinson(1993)); PPO2 (HortResearch; Kim et al. (2001)); GPO3 (HortResearch,Boss), APO; PPO3 and PPO7 (Haruta et al. (1998).

In an embodiment, PPO2 comprises, consists of, or consists essentiallyof the amino acid sequence set forth in SEQ ID NO: 19. In an embodiment,GPO3 comprises, consists of, or consists essentially of the amino acidsequence set forth in SEQ ID NO: 21. In an embodiment, APO5 comprises,consists of, or consists essentially of the amino acid sequence setforth in SEQ ID NO: 23. In an embodiment, PSR7 comprises, consists of,or consists essentially of the amino acid sequence set forth in SEQ IDNO 25.

It is anticipated that some apple varieties or indeed even otherfruit-producing plant species may contain variants of PPO2, GPO3, APO5and PSR7 that may be targeted for reduction in activity in accordancewith the invention. Accordingly, the PPO isoenzyme may have an aminoacid sequence that possesses at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% sequence identity to SEQ ID NO: 19, 21, 23 or 25.Preferably such variant sequences are species or allelic variants of thePPO2, GPO3, APO5 or PSR7 sequence as set forth in SEQ ID NO: 19, 21, 23and 25, respectively.

The PPO isoenzyme may also be a fragment of a PPO isoenzyme as describedabove, the fragment possessing PPO activity. Such fragments may comprisee.g. at least 10, at least 20, at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least150, at least 200, at least 250, or at least 300 amino acids.

In an embodiment, the PPO isoenzyme described herein is a polypeptidethat retains at least some PPO activity of any one of the appleisoenzymes APOS, GPO3, PPO2 or pSR7 but differs in sequence from any oneof these by one or more amino acid insertions, deletions, orsubstitutions, particularly conservative amino acid substitutions. Asused herein, the expression “conservative amino acid substitutions”refers to the substitution of one amino acid for another at a givenlocation in the polypeptide, where the substitution occurs withoutsubstantial loss of the relevant function. Conservative substitutionsgenerally involve substitution of an amino acid residue with anotheramino acid residue on the basis of relative similarity of side-chainsubstituents, for example, their size, charge, hydrophobicity,hydrophilicity, and the like. Specific, non-limiting examples of aconservative substitution include the following examples:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

As used herein, the term “polypeptide” encompasses any chain ofnaturally or non-naturally occurring amino acids (either D- or L-aminoacids), regardless of length (e.g., at least 5, 6, 8, 10, 12, 14, 16,18, 20, 25, 30, 40, 50, 100 or more amino acids) or post-translationalmodification (e.g., glycosylation or phosphorylation) or the presence ofe.g. one or more non-amino acyl groups (for example, sugar, lipid, etc.)covalently linked to the peptide, and includes, for example, naturalproteins, synthetic or recombinant polypeptides and peptides, hybridmolecules, peptoids, peptidomimetics, etc. As used herein, the terms“polypeptide”, “peptide” and “protein” may be used interchangeably.

Other apple PPO isoenzymes may be identified for use in the context ofthe invention. As disclosed herein, a degenerate primer approach wasused to identify novel PPO gene sequences. Other approaches to identifynovel PPO isoenzymes are known in the art include, but not limited to,use of degenerate primers to screen an expression library from plantsthat produce fruit susceptible to browning; and the use ofbioinformatics to virtually identify other PPO genes. Following theidentification of a candidate PPO gene, it may be disrupted in a fruitproducing plant and the produced fruit may be assessed for alteredfruit-browning properties, with any of the approaches disclosed herein.

Other approaches to identify PPO isoenzymes include, without limitation,screening cDNA, genomic or bac libraries with PPO probes from apple orother species. Alternatively, sequences could be identified in the applegenome sequence soon to be published (IASMA—Istituto Agrario San Micheleall'Adige).

When the invention is practiced in a pear plant, non-limiting examplesof PPO isoenzymes that may be targeted for reduced activity includewithout limitation PPOs encoded by PearGB [SEQ ID NO:66]; Pearl [SEQ IDNO:67] and Pear2 [SEQ ID NO:68], or fragments or variants thereof asdescribed above.

Reduction of PPO Activity or Expression

PPO expression and/or activity in genetically modified plants of thepresent invention may be reduced by any method that results in reducedactivity of at least two PPO isoenzymes in the plant. This may beachieved by e.g. by altering PPO at the DNA, mRNA and/or protein levels.

As used herein, “activity” refers to the biochemical reaction of anenzyme with its cognate substrate. In the context of the invention,reduced PPO activity may result from reduced protein levels of a PPOisoenzyme and/or the reduced rate at which a PPO isoenzyme catalyzes itsreaction with a substrate.

Total PPO activity may be determined, without limitation, by using thepolyphenol oxidase specific activity assay of Broothaerts et al (2000),or a modification thereof, for example, the assay adapted for use inmicrotitre plate format. PPO specific activity may be expressed in termsof U/mg protein. Substrates that may be used in the assay to determinePPO specific activity are known in the art, and include, withoutlimitation, 4-methyl catechol.

In one embodiment, PPO expression and/or activity may be altered bytargeting genomic PPO genes. For example, the endogenous PPO gene may bealtered by, without limitation, knocking-out one or more PPO genes; orknocking-in a heterologous DNA to disrupt one or more PPO genes. Theskilled person would understand that these approaches may be applied tothe coding sequences, the promoter or other regulatory elementsnecessary for gene transcription.

In another embodiment, PPO expression and/or activity may be altered bytargeting PPO mRNA transcripts. In this regard, levels of PPO mRNAtranscripts may be reduced by methods known in the art including, butnot limited to, co-suppression, antisense expression, small hair pin(shRNA) expression (Zhao et al.), interfering RNA (RNAi) expression(Matzke et al.), double stranded (dsRNA) expression (Karkare et al.),inverted repeat dsRNA expression (Otani et al.), micro interfering RNA(miRNA) (Willmann and Poethig, or Pikaard), simultaneous expression ofsense and antisense sequences (Karkare et al.), or a combinationthereof, targeted at least two PPO isoenzymes encoding genes.

The phenomenon of co-suppression in plants relates to the introductionof transgenic copies of a gene resulting in reduced expression of thetransgene as well as the endogenous gene (Napoli et al (1990); van derKrol et al. (1990). The observed effect depends on sequence identitybetween the transgene and the endogenous gene.

RNA interference/silencing relates to the silencing of genes by theintroduction of double stranded RNA. RNA is both an initiator and targetin the process (Fire et al, (1998); Lindbo et al, (1993); Montgomery etal, (1998)). This mechanism targets RNA from viruses and transposons andalso plays a role in regulating development and genome maintenance.Briefly, double stranded RNA is cleaved by the enzyme dicer resulting inshort fragments of 21-23 bp (siRNA). One of the two strands of eachfragment is incorporated into the RNA-induced silencing complex (RISC).The RISC associated RNA strand pairs with mRNA and induces cleavage ofthe mRNA. Alternatively, RISC associated RNA strand pairs with genomicDNA resulting in epigenetic changes that affect gene transcription.Micro RNA (miRNA) is a type of RNA transcribed from the genome itselfand works in a similar way. Similarly, shRNA may be cleaved by dicer andassociate with RISC resulting in mRNA cleavage.

Antisense suppression of gene expression does not involve the catalysisof mRNA degradation, but instead involves single-stranded RNA fragmentsbinding to mRNA and blocking protein translation.

Both antisense and sense suppression are mediated by silencing RNAs(sRNAs) produced from either a sense-antisense hybrid or double strandedRNA (dsRNA) generated by an RNA-dependant RNA polymerase (Jorgensen2006). Majors classes or sRNAs include short-interfering RNAs (siRNAs)and microRNAs (miRNAs) which differ in their biosynthesis.

Processing of dsRNA precursors by Dicer-Like complexes yields21-nucleotide siRNAs and miRNAs guide cleavage of target transcriptsfrom within RNA-induced silencing complexes (RISC).

Preferably PPO expression may be suppressed using an synthetic gene oran unrelated gene that contained about 21 bp regions of high homology(preferably 100% homology) to the PPO gene.

See, for example, Jorgensen R A, Doetsch N, Muller A, Que Q, Gendler, Kand Napoli C A (2006) A paragenetic perspective on integration of RNAsilencing into the epigenome and in the biology of higher plants. ColdSpring Harb. Symp. Quant. Biol. 71:481-485.

For a review, see for example, Ossowski S, Schwab R and Weigel D (2008)Gene silencing in plants using artificial microRNAs and other smallRNAs. The Plant Journal 53:674-690.

In a further embodiment, PPO activity may be altered by targeting one ormore PPO isoenzymes at the protein level. For example, a PPO isoenzymeactivity may be altered by affecting the post-translational modificationof the enzyme; or by the introduction of a heterologous protein (e.g. amutated form of one or more PPO isoenzymes may be expressed such that itassociates with the wildtype PPO isoenzyme and alters its activity; oran antibody that binds specifically to one or more PPO isoenzymes).

As used herein, “expression” or “expressing” refers to production of anydetectable level of a product encoded by the coding sequence. In thecontext of the invention, reduced PPO expression may result from reducedtranscription of a PPO gene or from reduced translation of PPO mRNAtranscripts.

In one embodiment, a genetically modified fruit producing plant of theinvention comprises, stably integrated into its genome a first nucleicacid molecule heterologous to the plant, the presence of the firstnucleic acid molecule reducing expression of a first PPO isoenzyme; anda second nucleic acid molecule heterologous to the plant, the presenceof the second nucleic acid molecule in the plant reducing expression ofa second PPO isoenzyme.

In one embodiment, a genetically modified plant of the present inventionmay further comprise a third nucleic acid molecule heterologous to theplant, the presence of said third nucleic acid molecule reducingexpression of a third PPO isoenzyme.

In another embodiment, the genetically modified plant of the presentinvention may further comprise a fourth nucleic acid moleculeheterologous to the plant, the presence of the fourth nucleic acidmolecule reducing expression of a fourth PPO isoenzyme.

If a plant expresses additional PPO isoenzymes, additional heterologousnucleic acid molecules (e.g. fifth, sixth, seventh and eighthheterologous nucleic acid molecules) may also be used to reduceexpression of such PPO isoenzymes.

A nucleic acid molecule that reduces the expression and/or activity ofany PPO isoenzyme may be used in the context of the invention. Forinstance, suitable apple PPO isoenzymes that may be targeted in appleplants to reduce browning in the fruit of the plant include thosedescribed above.

Further, a nucleic acid sequence comprising a nucleic acid sequence thatis at least 65%, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% identical to the nucleic acid sequence of any known appleor other fruit PPO isoenzyme may be suitable for use in the context ofthe invention.

Fragments of nucleic acid sequences encoding fruit PPO isoenzymes may beused. Such fragments may have lengths of at least 20, at least 50, atleast 100, at least 150, at least 200, at least 300 or at least 400contiguous nucleotides of a nucleic acid sequence encoding a PPO.Alternatively such fragments may have a minimum length of at least 20,at least 25, at least 30, at least 35, at least 40, at least 45, or atleast 50 contiguous nucleotides and a maximum length less than 3000,less than 2000, less than 1750, less than 1500, less than 1250, lessthan 1000, less than 750 or less than 500 contiguous nucleotides or anycombination of such minimum and maximum lengths of a nucleic acidsequence encoding a PPO.

In one aspect, a nucleic acid molecule may comprise PPO2 (SEQ ID NO:18); GPO3 (SEQ ID NO: 20); APOS (SEQ ID NO: 22); pSR7 (SEQ ID NO: 24); aspecies variant thereof, an allelic variant thereof; a non-naturalvariant thereof; or a fragment thereof.

In one embodiment, a fragment of PPO2 as set forth in SEQ ID NO: 26; afragment of GPO3 as set forth in SEQ ID NO: 27; a fragment of APO5 asset forth in SEQ ID NO: 28; and a fragment of pSR7 as set forth in SEQID NO: 29 may be suitable for use in the context of the invention.

In another embodiment, a fragment of PPO2 as set forth in SEQ ID NO: 30;a fragment of GPO3 as set forth in SEQ ID NO: 31; a fragment of APO5 asset forth in SEQ ID NO: 32; and a fragment of pSR7 as set forth in SEQID NO: 33 may be suitable for use in the context of the invention.

The gene fragments PPO2, GPO3, APO5, and pSR7 described herein aremerely illustrative. Gene fragments suitable for use in the context ofthe invention may differ in sequence, in length and in location relativeto those noted above may be suitable for use in the context of theinvention.

For example, a gene fragment (such as a fragment of e.g. PPO2, GPO3,APOS, or pSR7) may comprise at least 20, at least 40, at least 60, atleast 80, at least 100, at least 150, at least 200, at least 150, atleast 300, at least 350, at least 400, at least 450 or at least 500contiguous nucleotides of said genes. A gene fragment of PPO2, GPO3,APO5 and/or pSR7 may be 5′ or 3′ of the fragments of those genesdisclosed herein.

Nucleic acid molecules that are substantially identical to the PPO2,GPO3, APOS, and pSR7 genes disclosed herein, may also be used in thecontext of the invention. As used herein, one nucleic acid molecule maybe “substantially identical” to another if the two molecules have atleast 60%, at least 70%, at least 80%, at least 82.5%, at least 85%, atleast 87.5%, at least 90%, at least 92.5%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% sequence identity. In oneembodiment, the two nucleic acid molecules each comprise at least 20identical contiguous nucleotides.

In various embodiments of the invention, the at least two heterologousnucleic acid molecules are selected from:

at least 20, at least 50, at least 100, at least 150, at least 200, atleast 300 or at least 400 contiguous nucleotides of a nucleic acidsequence possessing at least 80%, at least 90% or 100% sequence identityto the nucleic acid sequence set forth in SEQ ID NO: 18;

at least 20, at least 50, at least 100, at least 150, at least 200, atleast 300 or at least 400 contiguous nucleotides of a nucleic acidsequence possessing at least 80%, at least 90% or 100% sequence identityto the nucleic acid sequence set forth in SEQ ID NO: 20;

at least 20, at least 50, at least 100, at least 150, at least 200, atleast 300 or at least 400 contiguous nucleotides of a nucleic acidsequence possessing at least 80%, at least 90% or 100% sequence identityto the nucleic acid sequence set forth in SEQ ID NO: 22; and

at least 20, at least 50, at least 100, at least 150, at least 200, atleast 300 or at least 400 contiguous nucleotides of a nucleic acidsequence possessing at least 80%, at least 90% or 100% sequence identityto the nucleic sequence set forth in SEQ ID NO: 24.

In other embodiments of the invention, the at least two heterologousnucleic acid molecules are selected from:

a nucleic acid molecule with a minimum length of at least 20, at least25, at least 30, at least 35, at least 40, at least 45, or at least 50contiguous nucleotides and a maximum length less than 1750, less than1500, less than 1250, less than 1000, less than 750 or less than 500contiguous nucleotides or any combination of such minimum and maximumlengths of a nucleic acid sequence possessing at least 80%, at least 90%or 100% sequence identity to the nucleic acid sequence set forth in SEQID NO: 18;

a nucleic acid molecule with a minimum length of at least 20, at least25, at least 30, at least 35, at least 40, at least 45, or at least 50contiguous nucleotides and a maximum length less than 1750, less than1500, less than 1250, less than 1000, less than 750 or less than 500contiguous nucleotides or any combination of such minimum and maximumlengths of a nucleic acid sequence possessing at least 80%, at least 90%or 100% sequence identity to the nucleic acid sequence set forth in SEQID NO: 20;

a nucleic acid molecule with a minimum length of at least 20, at least25, at least 30, at least 35, at least 40, at least 45, or at least 50contiguous nucleotides and a maximum length less than 1750, less than1500, less than 1250, less than 1000, less than 750 or less than 500contiguous nucleotides or any combination of such minimum and maximumlengths of a nucleic acid sequence possessing at least 80%, at least 90%or 100% sequence identity to the nucleic acid sequence set forth in SEQID NO: 22; and

a nucleic acid molecule with a minimum length of at least 20, at least25, at least 30, at least 35, at least 40, at least 45, or at least 50contiguous nucleotides and a maximum length less than 1750, less than1500, less than 1250, less than 1000, less than 750 or less than 500contiguous nucleotides or any combination of such minimum and maximumlengths of a nucleic acid sequence possessing at least 80%, at least 90%or 100% sequence identity to the nucleic sequence set forth in SEQ IDNO: 24.

The term “identity” refers to sequence similarity between twopolypeptide or polynucleotide molecules. Identity can be determined bycomparing each position in the aligned sequences. A degree of identitybetween amino acid or nucleic acid sequences is a function of the numberof identical or matching amino acids or nucleic acids at positionsshared by the sequences, for example, over a specified region. Optimalalignment of sequences for comparisons of identity may be conductedusing a variety of algorithms, as are known in the art, including theClustal W™ program, available at http://clustalw.genome.ad.jp, the localhomology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482,the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48:443, the search for similarity method of Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85:2444, and the computerisedimplementations of these algorithms (such as GAP, BESTFIT, FASTA andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, Madison, Wis., U.S.A.). Sequence identity may also be determinedusing the BLAST algorithm (e.g. BLASTn and BLASTp), described inAltschul et al., 1990, J. Mol. Biol. 215:403-10 (using the publisheddefault settings). Software for performing BLAST analysis is availablethrough the National Center for Biotechnology Information (through theInternet at http://www.ncbi.nlm.nih.gov/). For instance, sequenceidentity between two nucleic acid sequences can be determined using theBLASTn algorithm at the following default settings: expect threshold 10;word size 11; match/mismatch scores 2,-3; gap costs existence 5,extension 2. Sequence identity between two amino acid sequences may bedetermined using the BLASTp algorithm at the following default settings:expect threshold 10; word size 3; matrix BLOSUM 62; gap costs existence11, extension 1. In another embodiment, the person skilled in the artcan readily and properly align any given sequence and deduce sequenceidentity/homology by mere visual inspection.

In the alternative, two nucleic acid sequences encoding PPO isoenzymesmay be substantially complementary (or are homologues/have identity) ifthe two sequences hybridize to each other under moderately stringent, orpreferably stringent, conditions. Hybridization to filter-boundsequences under moderately stringent conditions may, for example, beperformed in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al.(eds), 1989, Current Protocols in Molecular Biology, Vol. 1, GreenPublishing Associates, Inc., and John Wiley & Sons, Inc., New York, atp. 2.10.3). Alternatively, hybridization to filter-bound sequences understringent conditions may, for example, be performed in 0.5 M NaHPO₄, 7%SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (seeAusubel, et al. (eds), 1989, supra). Hybridization conditions may bemodified in accordance with known methods depending on the sequence ofinterest (see Tijssen, 1993, Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, New York). Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint for the specific sequence at a defined ionic strength and pH.

The first, second, third and/or fourth or additional nucleic acidmolecules may be present in a single genetic construct or in multipleconstructs. In one embodiment, the first, second, third and/or fourth oradditional nucleic acid molecules may be arranged in the senseorientation relative to a promoter. In another embodiment, the first,second, third and/or fourth or additional nucleic acid molecules may bearranged in the anti-sense orientation relative to a promoter. In afurther embodiment, a genetic construct may comprise at least twonucleic acid molecules in both the sense and anti-sense orientations,relative to a promoter. A genetic construct comprising nucleic acids inboth the sense and anti-sense orientations may result in mRNAtranscripts capable of forming stem-loop structures.

One or more of the nucleic acid molecules may be under transcriptionalcontrol of the same promoter.

A genetic construct comprising nucleic acids in both orientationsrelative to a promoter may further comprise a spacer to separate thenucleic acid molecules in sense orientation and those in the anti-senseorientation. As used herein, a “spacer” may comprise at least 2, atleast 5, at least 10, at least 20, at least 30, at least 40, at least50, at least 75, at least 100, at least 150, or at least 200nucleotides. In one embodiment, the spacer may be an intron, such as anintron from a PPO gene.

In the context of the present invention, the nucleic acid molecules maycomprise nucleic acid that is heterologous to the plant in which PPOactivity is reduced. As used herein, “heterologous”, “foreign” and“exogenous” DNA and RNA are used interchangeably and refer to DNA or RNAthat does not occur naturally as part of the plant genome in which it ispresent or which is found in a location or locations in the genome thatdiffer from that in which it occurs in nature. Thus, heterologous orforeign DNA or RNA is nucleic acid that is not normally found in thehost genome in an identical context (i.e. linked to identical 5′ and 3′sequences). In one aspect, heterologous DNA may be the same as the hostDNA but introduced into a different place in the host genome and/or hasbeen modified by methods known in the art, where the modificationsinclude, but are not limited to, insertion in a vector, linked to aforeign promoter and/or other regulatory elements, or repeated atmultiple copies. In another aspect, heterologous DNA may be from adifferent organism, a different species, a different genus or adifferent kingdom, as the host DNA. Further, the heterologous DNA may bea transgene. As used herein, “transgene” refers to a segment of DNAcontaining a gene sequence that has been isolated from one organism andintroduced into a different organism.

As used herein, “nucleotide sequence”, “polynucleotide sequence”,“nucleic acid” or “nucleic acid molecule” may refer to a polymer of DNAor RNA which can be single or double stranded and optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. “Nucleic acid”, “nucleic acidsequence”, “polynucleotide sequence” or “nucleic acid molecule” mayencompass genes, cDNA, DNA and RNA encoded by a gene. Nucleic acids,nucleic acid sequences, polynucleotide sequence and nucleic acidmolecule may comprise at least 3, at least 10, at least 100, at least1000, at least 5000, or at least 10000 nucleotides or base pairs.

As used herein, a “fragment”, a “fragment thereof”, “gene fragment” or a“gene fragment thereof” refers to a portion of a “nucleotide sequence”,“polynucleotide sequence”, “nucleic acid” or “nucleic acid molecule”that may still reduce total PPO gene expression and/or fruit browning.In one embodiment, the fragment comprises at least 20, at least 40, atleast 60, at least 80, at least 100, at least 150, at least 200, atleast 150, at least 300, at least 350, at least 400, at least 450 or atleast 500 contiguous nucleotides.

As used herein, a “non-natural variant” refers to nucleic acid sequencesnative to an organism but comprising modifications to one or more of itsnucleotides. Nucleic acids may be modified by any chemical and/orbiological means known in the art including, but not limited to,reaction with any known chemicals such as alkylating agents, browningsugars, etc; conjugation to a linking group (e.g. PEG); methylation;oxidation; ionizing radiation; or the action of chemical carcinogens.Such nucleic acid modifications may occur during synthesis or processingor following treatment with chemical reagents known in the art.

As used herein, a “species variant” refers to an alternate form of thesame PPO gene as found in different species of the same genus. The termmay also refer to an alternate form of the same PPO gene as found indifferent varieties of the same species, for example, of a plant.

As used herein, an “allelic variant” refers to an alternate form of thesame gene at a specific location of the genome.

As used herein, “wildtype” may refer to a plant or plant material thatwas not transformed with a nucleic acid molecule or construct, asdescribed herein. A “wildtype” may also refer to a plant or plantmaterial in which total PPO activity was not reduced from a reduction inactivity of at least two PPO isoenzymes.

The person skilled in the art will also readily understand that althoughin the foregoing illustrative examples partial PPO coding sequences wereused to construct the PPO suppression transgene, complete PPO codingsequences, alternative PPO coding sequences, 5′UTR and/or 3′UTR, ormutated derivatives of these sequences can also be used.

The skilled person would appreciate that the reduction in activity of atleast two PPO isoenzymes may not be limited by the number of differentnucleic acid molecules introduced into a plant or plant cell. In oneembodiment, one nucleic acid molecule may target one or more PPOisoenzyme genes. For example, one nucleic acid molecule may target atleast one, at least two, at least three, or more PPO isoenzyme genes. Inanother example, 3 gene segments may be used to effect suppression of 4PPO isoenzyme gene targets: 1 segment specific for PPO2, 1 segmentspecific for pSR7, and 1 segment capable of targeting both APOS and GPO3(for example, due to microhomology). In another embodiment, one or morenucleic acid molecules may be used to target one PPO isoenzyme gene. Ina further embodiment, one nucleic acid molecule may target one PPOisoenzyme gene.

The maximum number of nucleic acid molecules that may be used in thecontext of the invention may be limited only by the maximum size of theconstruct that may be delivered to a target plant or plant cell using agiven transformation method.

The skilled person would also appreciate that a nucleic acid moleculecomprising the sequence of a PPO gene promoter and/or other regulatoryelements may be used in the context of the invention. In an embodiment,a heterologous nucleic acid molecule comprising sequences of a PPO genepromoter and/or regulatory element may be used to bias the cellularmachinery away from an endogenous PPO gene promoter thus resulting inreduced PPO gene expression.

Suppression Construct

A construct of the invention comprising a first, second, third and/orfourth nucleic acid molecule may further comprise a promoter and otherregulatory elements, for example, an enhancer, a silencer, apolyadenylation site, a transcription terminator, a selectable marker ora screenable marker.

As used herein, a “vector” or a “construct” may refer to any recombinantpolynucleotide molecule such as a plasmid, cosmid, virus, vector,autonomously replicating polynucleotide molecule, phage, or linear orcircular single-stranded or double-stranded DNA or RNA polynucleotidemolecule, derived from any source. A “vector” or a “construct” maycomprise a promoter, a polyadenylation site, an enhancer or silencer anda transcription terminator, in addition to a nucleotide sequenceencoding a gene or a gene fragment of interest. As used herein, a“transformation vector” may refer to a vector used in the transformationof, or in the introduction of DNA into, cells, plants or plantmaterials.

As used herein, a “promoter” refers to a nucleotide sequence thatdirects the initiation and rate of transcription of a coding sequence(reviewed in Roeder, Trends Biochem Sci, 16: 402, 1991). The promotercontains the site at which RNA polymerase binds and also contains sitesfor the binding of other regulatory elements (such as transcriptionfactors). Promoters may be naturally occurring or synthetic (see Datlaet al. Biotech Ann. Rev 3:269, 1997 for review of plant promoters).Further, promoters may be species specific (for example, active only inB. napus); tissue specific (for example, the napin, phaseolin, zein,globulin, dlec2, γ-kafirin seed specific promoters); developmentallyspecific (for example, active only during embryogenesis); constitutive(for example maize ubiquitin, rice ubiquitin, rice actin, Arabidopsisactin, sugarcane bacilliform virus, CsVMV and CaMV 35S, Arabidopsispolyubiquitin, Solanum bulbocastanum polyubiquitin, Agrobacteriumtumefaciens-derived nopaline synthase, octopine synthase, and mannopinesynthase gene promoters); or inducible (for example the stilbenesynthase promoter and promoters induced by light, heat, cold, drought,wounding, hormones, stress and chemicals). A promoter includes a minimalpromoter that is a short DNA sequence comprised of a TATA box or an Inrelement, and other sequences that serve to specify the site oftranscription initiation, to which regulatory elements are added forcontrol of expression. A promoter may also refer to a nucleotidesequence that includes a minimal promoter plus DNA elements thatregulates the expression of a coding sequence, such as enhancers andsilencers. Thus in one aspect, the expression of the constructs of thepresent invention may be regulated by selecting a species specific, atissue specific, a development specific or an inducible promoter.

Enhancers and silencers are DNA elements that affect transcription of alinked promoter positively or negatively, respectively (reviewed inBlackwood and Kadonaga, Science, 281: 61, 1998).

Polyadenylation site refers to a DNA sequence that signals the RNAtranscription machinery to add a series of the nucleotide A at about 30bp downstream from the polyadenylation site.

Transcription terminators are DNA sequences that signal the terminationof transcription. Transcription terminators are known in the art. Thetranscription terminator may be derived from Agrobacterium tumefaciens,such as those isolated from the nopaline synthase, mannopine synthase,octopine synthase genes and other open reading frame from Ti plasmids.Other terminators may include, without limitation, those isolated fromCaMV and other DNA viruses, dlec2, zein, phaseolin, lipase, osmotin,peroxidase, PinII and ubiquitin genes, for example, from Solanumtuberosum.

In the context of the invention, the nucleic acid construct may furthercomprise a selectable marker. Selectable markers may be used to selectfor plants or plant cells that contain the exogenous genetic material.The exogenous genetic material may include, but is not limited to, anenzyme that confers resistance to an agent such as a herbicide or anantibiotic, or a protein that reports the presence of the construct.

Numerous plant selectable marker systems are known in the art and areconsistent with this invention. The following review article illustratesthese well known systems: Miki and McHugh; Journal of Biotechnology 107:193-232; Selectable marker genes in transgenic plants: applications,alternatives and biosafety (2004).

Examples of a selectable marker include, but are not limited to, a neogene, which codes for kanamycin resistance and can be selected for usingkanamycin, NptII, G418, hpt etc.; an amp resistance gene for selectionwith the antibiotic ampicillin; an hygromycinR gene for hygromycinresistance; a BAR gene (encoding phosphinothricin acetyl transferase)which codes for bialaphos resistance including those described inWO/2008/070845; a mutant EPSP synthase gene, aadA, which encodesglyphosate resistance; a nitrilase gene, which confers resistance tobromoxynil; a mutant acetolactate synthase gene (ALS), which confersimidazolinone or sulphonylurea resistance, ALS, and a methotrexateresistant DHFR gene.

Further, screenable markers that may be used in the context of theinvention include, but are not limited to, a) β-glucuronidase or uidAgene (GUS), which encodes an enzyme for which various chromogenicsubstrates are known, green fluorescent protein (GFP), and luciferase(LUX).

The size or length of the nucleic acid construct or elements thereof,are not limited to the specific embodiments described herein. Forexample, the skilled person would appreciate that the size of atransgene element may be defined instead by transgene element function;and that the promoter element may be determined instead as one that wascapable of driving transcription at a sufficient level and in thedesired tissues. Similarly, the stem loop structure formed by the mRNAtranscribed by a nucleic acid construct of the invention, may comprise anumber of gene segments which may vary in length. For example, the stemloop may comprise 3 gene segments of about 21-30 basepairs each, inaddition to a spacer, such as an intron (126 bp plus intron).

The skilled person would appreciate that the size of the gene segmentsmay be established by the sum of the element sizes combined and maydepend on the transformation method used to deliver the transgene intothe target organism. For example, each transformation method(Agrobacterium, biolistics, VIGS-based delivery systems) may be limitedto theoretical maximum transgene sizes.

Plant Transformation

The present invention is not limited to any particular method fortransforming plant cells. Methods for introducing nucleic acids intocells (also referred to herein as “transformation”) are known in the artand include, but are not limited to: Viral methods (Clapp. ClinPerinatol, 20: 155-168, 1993; Lu et al. J Exp Med, 178: 2089-2096, 1993;Eglitis and Anderson. Biotechniques, 6: 608-614, 1988; Eglitis et al,Avd Exp Med Biol, 241: 19-27, 1988); physical methods such asmicroinjection (Capecchi. Cell, 22: 479-488, 1980), electroporation(Wong and Neumann. Biochim Biophys Res Commun, 107: 584-587, 1982; Frommet al, Proc Natl Acad Sci USA, 82: 5824-5828, 1985; U.S. Pat. No.5,384,253) and the gene gun (Johnston and Tang. Methods Cell Biol, 43:353-365, 1994; Fynan et al. Proc Natl Acad Sci USA, 90: 11478-11482,1993); chemical methods (Graham and van der Eb. Virology, 54: 536-539,1973; Zatloukal et al. Ann NY Acad Sci, 660: 136-153, 1992); andreceptor mediated methods (Curiel et al. Proc Natl Acad Sci USA, 88:8850-8854, 1991; Curiel et al. Hum Gen Ther, 3: 147-154, 1992; Wagner etal. Proc Natl Acad Sci USA, 89: 6099-6103, 1992).

The introduction of DNA into plant cells by Agrobacterium mediatedtransfer is well known to those skilled in the art. If, for example, theTi or Ri plasmids are used for the transformation of the plant cell, atleast the right border, although more often both the right and the leftborder of the T-DNA contained in the Ti or Ri plasmid must be linked tothe genes to be inserted as flanking region. If agrobacteria are usedfor the transformation, the DNA to be integrated must be cloned intospecial plasmids and specifically either into an intermediate or abinary vector. The intermediate vectors may be integrated into the Ti orRi plasmid of the agrobacteria by homologous recombination due tosequences, which are homologous to sequences in the T-DNA. This alsocontains the vir-region, which is required for T-DNA transfer.Intermediate vectors cannot replicate in agrobacteria. The intermediatevector can be transferred to Agrobacterium tumefaciens by means of ahelper plasmid (conjugation). Binary vectors are able to replicate in E.coli as well as in agrobacteria. They contain a selection marker geneand a linker or polylinker framed by the right and left T-DNA borderregion. They can be transformed directly into agrobacteria. Theagrobacterium acting as host cell should contain a plasmid carrying avir-region. The vir-region is required for the transfer of the T-DNAinto the plant cell. Additional T-DNA may be present. Such a transformedagrobacterium is used for the transformation of plant cells. The use ofT-DNA for the transformation of plant cells has been intensively studiedand has been adequately described in standard review articles andmanuals on plant transformation. Plant explants cultivated for thispurpose with Agrobacterium tumefaciens or Agrobacterium rhizogenes canbe used for the transfer of DNA into the plant cell.

Although Agrobacterium tumefaciens LBA4404 transformation has beendescribed, a person skilled in the art will readily understand that anyother suitable method of DNA transfer into plant may be used.

Another method for introducing DNA into plant cells is by biolistics.This method involves the bombardment of plant cells with microscopicparticles (such as gold or tungsten particles) coated with DNA. Theparticles are rapidly accelerated, typically by gas or electricaldischarge, through the cell wall and membranes, whereby the DNA isreleased into the cell and incorporated into the genome of the cell.This method is used for transformation of many crops, including corn,wheat, barley, rice, woody tree species and others. Biolisticbombardment has been proven effective in transfecting a wide variety ofanimal tissues as well as in both eukaryotic and prokaryotic microbes,mitochondria, and microbial and plant chloroplasts (Johnston. Nature,346: 776-777, 1990; Klein et al. Bio/Technol, 10: 286-291, 1992;Pecorino and Lo. Curr Biol, 2: 30-32, 1992; Jiao et al, Bio/Technol, 11:497-502, 1993).

Another method for introducing DNA into plant cells is byelectroporation. This method involves a pulse of high voltage applied toprotoplasts/cells/tissues resulting in transient pores in the plasmamembrane which facilitates the uptake of foreign DNA. The foreign DNAenter through the holes into the cytoplasm and then to the nucleus.

Plant cells may be transformed by liposome mediated gene transfer. Thismethod refers to the use of liposomes, circular lipid molecules with anaqueous interior, to deliver nucleic acids into cells. Liposomesencapsulate DNA fragments and then adhere to the cell membranes and fusewith them to transfer DNA fragments. Thus, the DNA enters the cell andthen to the nucleus.

Other well-known methods for transforming plant cells which areconsistent with the present invention include, but are not limited to,pollen transformation (See University of Toledo 1993 U.S. Pat. No.5,177,010); Whiskers technology (See U.S. Pat. Nos. 5,464,765 and5,302,523).

The nucleic acid constructs of the present invention may be introducedinto plant protoplasts. Plant protoplasts are cells in which its cellwall is completely or partially removed using either mechanical orenzymatic means, and may be transformed with known methods including,calcium phosphate based precipitation, polyethylene glycol treatment andelectroporation (see for example Potrykus et al., Mol. Gen. Genet., 199:183, 1985; Marcotte et al., Nature, 335: 454, 1988). Polyethylene glycol(PEG) is a polymer of ethylene oxide. It is widely used as a polymericgene carrier to induce DNA uptake into plant protoplasts. PEG may beused in combination with divalent cations to precipitate DNA and effectcellular uptake. Alternatively, PEG may be complexed with otherpolymers, such as poly(ethylene imine) and poly L lysine.

A nucleic acid molecule of the present invention may also be targetedinto the genome of a plant cell by a number of methods including, butnot limited to, targeting recombination, homologous recombination andsite-specific recombination (see review Baszcynski et al. TransgenicPlants, 157: 157-178, 2003 for review of site-specific recombinationsystems in plants). Homologous recombination and gene targeting inplants (reviewed in Reiss. International Review of Cytology, 228:85-139, 2003) and mammalian cells (reviewed in Sorrell and Kolb.Biotechnology Advances, 23: 431-469, 2005) are known in the art.

As used herein, “targeted recombination” refers to integration of anucleic acid construct into a site on the genome, where the integrationis facilitated by a construct comprising sequences corresponding to thesite of integration.

Homologous recombination relies on sequence identity between a piece ofDNA that is introduced into a cell and the cell's genome. Homologousrecombination is an extremely rare event in higher eukaryotes. However,the frequency of homologous recombination may be increased withstrategies involving the introduction of DNA double-strand breaks,triplex forming oligonucleotides or adeno-associated virus.

As used herein, “site-specific recombination” refers to the enzymaticrecombination that occurs when at least two discrete DNA sequencesinteract to combine into a single nucleic acid sequence in the presenceof the enzyme. Site-specific recombination relies on enzymes such asrecombinases, transposases and integrases, which catalyse DNA strandexchange between DNA molecules that have only limited sequence homology.Mechanisms of site specific recombination are known in the art (reviewedin Grindley et al. Annu Rev Biochem, 75: 567-605, 2006). The recognitionsites of site-specific recombinases (for example Cre and att sites) areusually 30-50 bp. The pairs of sites between which the recombinationoccurs are usually identical, but there are exceptions e.g. attP andattB of A integrase (Landy. Ann Rev Biochem, 58: 913-949, 1989).

The nucleic acid molecule becomes stably integrated into the plantgenome such that it is heritable to daughter cells in order thatsuccessive generations of plant cells have reduced PPO expression. Thismay involve the nucleic acid molecules of the present inventionintegrating, for instance integrating randomly, into the plant cellgenome. Alternatively, the nucleic acid molecules of the presentinvention may remain as exogenous, self-replicating DNA that isheritable to daughter cells. As used herein, exogenous, self-replicatingDNA that is heritable to daughter cells is also considered to be “stablyintegrated into the plant genome”.

Plant Culture

Plant cell culture techniques are known in the art (see for exampleFischer et al. Biotechnol Appl Biochem, 30: 109-112, 1999; Doran.Current Opinions in Biotechnology, 11: 199-204, 2000). The skilledperson would appreciate that the composition of the culture media, itspH and the incubating conditions, such as temperatures, aeration, CO₂levels, and light cycles, may vary depending on the type of cells.

Plant Selection

After transformation, plant cells may be sub-cloned to obtain clonalpopulations of cells. Methods of sub-cloning cells are known in the artand include, but are not limited to, limiting dilution of the pool oftransformed cells. For example, a construct of the invention maycomprise a selectable or screenable marker, as described herein. A celltransformed with a construct comprising a selection marker may be grownunder selective pressure to identify those that contain and/or expressthe construct.

Naturally, it could also be done without any selection marker, althoughthis would involve a fairly high screening expenditure. If marker-freegenetically modified plants are desired, there are also strategiesavailable to the person skilled in the art, which allow subsequentremoval of the marker gene, such as co-transformation andsequence-specific recombinases.

After preparing clonal populations of transgenic plant cells, the cellsmay be characterized and selected based on analysis at the level of DNA,RNA and protein. Preferably, transgenic plant cells in which the nucleicacid construct is stably integrated into the cell genome are selected.As used herein, “stably integrated” refers to the integration of geneticmaterial into the genome of the transgenic plant cell and remains partof the plant cell genome over time. The term “stably integrated” mayalso refer to the persistence of an exogenous replicating DNA that isheritable to daughter cells.

Stable integration of nucleic acid constructs may be influenced by anumber of factors including, but not limited to, the transformationmethod used and the vector containing the gene of interest. Thetransformation method determines which cell type can be targeted forstable integration. The type of vector used for stable integrationdefines the integration mechanism, the regulation of transgeneexpression and the selection conditions for stably expressing cells.After integration, the level and time of expression of the gene ofinterest may depend on the linked promoter and on the particularintegration site.

Plant Regeneration

Once the plant material is transformed, it may be regenerated intoplantlets or plants. Plant regeneration by tissue culture techniques iswell established. For example, plant regeneration from culturedprotoplasts is described in Evans et al, (1983); and Vasil I. R. (1986).Plants have been successfully micropropagated in vitro by organogenesisor somatic embryogenesis including, but not limited to, all majorspecies of sugarcane, sugar beet, cotton, fruit and other trees, legumesand vegetables. The methods for regeneration vary from species tospecies of plants, but generally a suspension of transformed protoplastscontaining copies of the heterologous gene is first provided. Callustissue is formed and shoots may be induced from callus and subsequentlyrooted. Alternatively, embryo formation may be induced from a protoplastsuspension. These embryos germinate to form mature plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. Shoots and roots normally develop simultaneously.Efficient regeneration will depend on the type of explant, thephysiological condition of the explant and physical and chemical mediaof the explant during culture, and on the history of the culture. Inanother alternative, plant material may be micrografted onto rootstocks.

Testing for Reduction of PPO Activity or Expression

Disruption of PPO genes, gene expression, or PPO enzymatic activity maybe confirmed by methods known in the art of molecular biology (see e.g.Maniatis et al., (1989)). For example, disruption of PPO genes may beassessed by PCR followed by Southern blot analysis. PPO mRNA levels may,for example, be measured by real time PCR, RT-PCR, Northern blotanalysis, micro-array gene analysis, and RNAse protection. PPO proteinlevels may, without limitation, be measured by enzyme activity assays,ELISA and Western blot analysis. PPO expression may be used as apredictor of reduced fruit browning. PPO enzymatic activity may beassessed biochemically or functionally.

For example, PPO activity may be measured biochemically by methods knownin the art including, but not limited to, the detection of productsformed by the enzyme in the presence of any number of heterologoussubstrates, for example, 4-methyl catechol. PPO activity may also bemeasured functionally, for example, by assessing its effects on fruitbrowning.

A genetically modified fruit-producing plant of the present inventionmay result in the reduction of total PPO activity in said plant or itsseed, seedling, progeny thereof, or produced fruit thereof, by at least57%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, or of at least 89%, relative to a wild type plant,seed, seedling, progeny thereof, or produced fruit thereof.

The skilled person would appreciate that the reduction in PPO activitymay vary depending on a number of factors including, but not limited to,the source of the PPO, the developmental stage of a plant or plantmaterial, the method of cultivation, the harvesting conditions, theexperimental conditions and variations thereof.

In one embodiment, the PPO specific activity of tissue culture leafmaterial produced from the genetically modified fruit-producing plant ofthe present invention may be reduced by at least 250, at least 500, atleast 750, at least 1000, at least 1250, at least 1500, at least 1750,at least 2000 or at least 2250 U/mg of protein as determined with thePPO specific activity assay of Broothaerts et al (2000), or amodification thereof, adapted for use in microtitre plate format;wherein the PPO specific activity of said wildtype of said plantaverages 2630 U/mg of protein.

In another embodiment, the PPO specific activity of immature fruitmaterial produced from the genetically modified fruit-producing plant ofthe present invention may be reduced by at least 10000, at least 15000,at least 20000, at least 25000, at least 30000, at least 35000, at least40000, at least 45000, at least 50000, at least 55000, at least 60000,at least 65000, or at least 70000 U/mg of protein as determined with thePPO specific activity assay of Broothaerts et al (2000), or amodification thereof, adapted for use in microtitre plate format;wherein the PPO specific activity of said wildtype of said plantaverages 75160 U/mg of protein.

A genetically modified fruit-producing plant of the present inventionmay result in the reduction of total PPO expression in said plant or itsseed, seedling, progeny thereof, or produced fruit thereof, by at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, relative to a wild type plant, seed, seedling, progeny thereof, orproduced fruit thereof.

Fruit Browning

As used herein, “reduced-browning” (or similar terminology) means thatwhen a fruit, such as an apple, is bruised, sliced, juiced or processedin a manner where cell wall destruction takes place, browning will bedetectably less than in a control. Any reduction in fruit browning (suchas a reduction in browning visible to the naked eye relative to acontrol) may be advantageous. In one embodiment, the rate of browning ofa fruit produced from a fruit-producing plant of the invention isreduced, relative to a control fruit. In another embodiment, the totalquantity or degree of browning of a fruit produced from afruit-producing plant of the invention is reduced, relative to a controlfruit.

For example, an individual eating an apple would likely find itadvantageous if the apple browned more slowly than a regular apple as inthe case of Ambrosia. If that same apple were cut to be packed into alunch or served on a fruit plate, then the apple would likely have tobrown very little over an extended period of time in order to beacceptable.

Also, a consumer may indicate that some browning is acceptable, as thisis the normal expectation. However, that same consumer would notpurchase an apple from the store if it had any form of bruise at thetime of purchase. So that consumer had already made considerableselection against apples that bruise.

Thus, industry would prefer an apple that did not suffer from bruising(which results in shrinkage) or browning (which results in lowerconsumer satisfaction). In other words, an apple that does not showvisible evidence of browning from mechanical damage such as bruising orslicing, may have commercial advantages.

Any detectable level of reduced browning that is detectable to the nakedeye may constitute a reduction in browning. Beyond this, reducedbrowning may be detected by a device, such as a chromameter, even if notvisible to the human eye. Reduction in fruit browning may be determinedin many ways, including, but not limited to, the difference inluminosity of a fruit tissue that is bruised in comparison to adjacent,unbruised tissue of the same fruit.

Fruit browning may be determined by known methods including, but notlimited to, spectroscopy (e.g. light absorption, laser-inducedfluorescence spectroscopy, time-delayed integration spectroscopy, largeaperture spectrometer); colorimetry (e.g. tristimulus, “spekol”spectrocolorimeter); and visual inspection/scoring. These approachesallow the detection of, among other parameters, changes in luminosityand color of bruised transgenic fruit in comparison to bruised controlfruit.

In one embodiment, a bruising apparatus may be used to deliver acontrolled bruise to fruit with minimal destruction to the tissue. Theparticular specifications of such an apparatus are not critical. What isimportant is that the apparatus permits fruit to be bruised in aconsistent manner so that the fruit may be used in controlled scientificstudies.

In one embodiment, browning may be measured by the change in luminosity(ΔL) or total change in color (SE) assays described herein.

Luminosity may be measured and expressed in terms of any number ofmodels including, but not limited to, the Hunter Lab color space or arelated implementation thereof (see, for example, Hunter (1948a and1948b)). In the Hunter Lab color space, L is a correlate of lightnesswhich ranges from 0-100, where 100 is white and 0 is black; a and b aretermed opponent color axes; a represents roughly Redness (positive) andGreenness (Negative), b is positive for yellow colors and negative forblue colors.

ΔL represents the change in luminosity between the unbruised apple(trt1) and the bruised apple flesh (trt2). A decrease in luminosity of2.0 units (ΔL=−2.0) or greater, using the Hunter color space model, isgenerally visible to the eye.

ΔE represents the change in total color between the unbruised apple andthe bruised apple flesh. LE is calculated from the formulae:ΔL=L _(trt 2) −L _(trt 1)Δa=L _(trt 2) −L _(trt 1)Δb=L _(trt 2) −L _(trt 1)ΔE=√{square root over ((ΔΔL ²+(ΔΔa ²+(Δb ²)}

A threshold for determining that a fruit has a reduced-browningphenotype is obtained when the ΔL (i.e. difference in luminosity betweena bruised area of a fruit and adjacent unbruised tissue) is less thanabout 0.5, less than about 1.0, less than about 1.5, less than about2.0, less than about 2.5, less than about 3.0, less than about 3.5, orless than about 4.0, using the Hunter color space model.

In one embodiment, in Golden Delicious apples, a decrease in luminosityof about 2.0 (ΔL=−2.0) is generally visible to the eye. For other applespecies and/or varieties the ΔL value that represents a visible bruisemay vary slightly from this depending on a number of factors, including,but not limited to, the natural flesh color of the apple (a and b coloropponents). In another embodiment, an apple may be detectablylow-browning to the naked eye if the decrease in luminosity is betweenabout 2.1 to 3.5 units.

Fruit browning may also be considered with respect to a wildtype orcontrol fruit and a test fruit produced from a reduced-browning plant ofthe invention. In an example, a control fruit and a test fruit producedfrom a reduced-browning plant of the invention are bruised in the same,or substantially the same way. Subsequently, the change in luminosity ΔLof each fruit is measured by detecting the luminosity at the site ofbruising in comparison to the luminosity at an adjacent, unbruised site,resulting in a control ΔL and a test sample ΔL. The test sample fruitmay be considered reduced-browning if the test sample ΔL is at least 1%,at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least12%, at least 13%, at least 14%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 60%, at least 70%, at least 80% or at least 90% less thanthe control ΔL.

The skilled person would appreciate that browning may vary depending ona number of factors including, but not limited to, the manner andambient conditions in which a fruit or plant material is bruised. Forexample, a fruit bruised at 2° C. may show different browningcharacteristics from a fruit bruised at 18° C., as detected by the eyeor by an instrument, such as a chromameter, or like devices.

The skilled person would also appreciate that fruit and/or other plantmaterial may be bruised in any number of ways. In one embodiment, fruitmay be bruised according to the Controlled Bruising Procedure asfollows.

Controlled Bruising Procedure

Summary

A controlled bruise is delivered to fruit (e.g. apples) in a controlledmanner with minimal destruction of tissue using an impact device asdescribed herein. Bruise response is reported as Change in Luminosity(ΔL), or Total Change in Colour (ΔE) between the bruised and non-bruisedtissue as measured using a Minolta Chroma Meter.

Equipment and Materials

The Impact Device

The Impact Device comprises the Impact Device itself, plus a shallowcontainer of glass beads into which the fruit is set, prior to beingbruised. The Impact Device comprises a wooden block with a roundedimpact surface that can be dropped from a consistent and adjustableheight. A bruise, as delivered by the Impact Device could,alternatively, be produced by dropping a marble or steel ball down atube, of a specific length, which is placed on the surface of the fruit.A shallow dish full of glass beads, into which the fruit is placed,provides a cushion to prevent damage to the underside of the fruitduring impact to the top side of the fruit.

The fruit is ideally bruised with minimum tissue damage. Excessiveimpact can damage tissue and produce a Change in Luminosity that isunrelated to the bruising.

Colour Meter

A colour meter, e.g. a Minolta colour meter, is used to measurebruising. The meter is calibrated according to manufacturer'sinstructions against a white background.

Procedure

Fruit is removed from storage and allowed to come to room temperaturefor 2 hours. Positions of the bruises are marked with a felt pen on thefruit skin. Each fruit is bruised 5× and allowed to sit at roomtemperature for 3 hours for the bruise to form. The fruit are peeledover the bruise areas without removing the pen marking or cutting deeplyinto the flesh of the fruit. Each peeled area is measured on thenon-bruised area adjacent to the bruise (trt 1) and directly on thebruised area (trt 2). Bruising, or Change in Luminosity (ΔL) iscalculated as: ΔL=trt2−trt1.

For further details concerning measurement of fruit bruising additionalinformation, see e.g. Rojas-Graü M. A. et al. (2006); Gnanasekharan V.et al. (1992); and McGuire R. G. (1992).

Uses of Fruit with Reduced Browning

Fruits, vegetables and/or plants of the present invention may havecommercial advantages. For example, reduced-browning produce that areeasily bruised would retain the appearance of undamaged fruit, thusretaining its commercial value. In other examples, the juice of fruitsand vegetables; or cut fruits and vegetables of the present inventionwould not require treatment with chemicals or other products to preventbrowning, thus retaining the flavour and wholesomeness of the product.Accordingly, the genetically modified fruit-producing plants of thepresent invention may have commercial advantages in the food industry,for example, grocery, baked goods, beverages and snack industries; inadvertisements and the advertising industry; in television programs(e.g. cooking shows); and in any business in which fruits and vegetablessusceptible to browning are featured or displayed.

The invention is further illustrated by the following, non-limitingexamples.

EXAMPLES

1. Reduction of a Single PPO Gene in Apples

In an illustrative example, the inventors have used a 250 bp fragment ofAP14 between the Cu binding sites (or that includes one of the Cubinding sites) in the antisense orientation under control of the CAMV35Spromoter (P_(CAMV35S)) and Nopaline Synthase terminator (T_(NOS)), whichfragment and was cloned into the binary vector pBINPLUS (van Engelen etal. 1995) to create the vector GEN-01. It was believed that the homologybetween all PPO sequences was sufficient that targeting any one of themwould result in sufficient reduction of total PPO expression.

AP14 is highly homologous to GPO3 at the 5′ end. Over the region of AP14cloned and sequenced, AP14 is 90% identical to GPO3 at the nucleotidesequence level, and 81% identical to GPO3 at the amino acid level (FIG.1). However, there is a change in the AP14 coding sequence thatgenerates a translational stop codon in the AP14 sequence, andapproximately 43 base pair (bp) downstream of this stop codon, there isa 7 base pair deletion in AP14, relative to GPO3, that would create aframeshift in the coding region. Therefore, it was thought that AP14 islikely a pseudogene copy of GPO3.

The inventors have also used a PPO suppression transgene in whichapproximately 800 bp of APO5 was placed in the antisense orientationunder control of the CAMV35S promoter (P_(CAMV35S)) and NopalineSynthase terminator (T_(NOS)) and cloned into the binary vector pBINPLUSto create the vector GEN-02, which was based on information that showedthat APO5 was the predominant species present in immature fruit.Additionally, Haruta et al. (1998) had reported reduced browning in leaftissue caused by antisense construct that carried PPO3, a homolog ofAPO5.

The inventors produced over 200 GEN-01 and 400 GEN-02 geneticallymodified lines and have identified no lines with significantly reducedPPO Activity or reduced browning phenotype.

2. Cloning and Sequencing of Apple PPO Genes

It was recognized that apple PPO activity was encoded by more than onePPO gene sequence although the sequences had not all been cloned and thefamily had not been clearly established.

Robinson (1993) identified pSR7 and pSR8 (later to be identified asPPO2), HortResearch found PPO2, GPO3, APO5 and pSR7 in their apple ESTlibrary (personal communication). Boss et al. (1995) identified APO5 andHaruta et al. (1998) identified PPO3 and PPO7 (APO5 homologs). Kim etal. (2001) identified PPO2. Boss also identified GPO3 (unpublished).

The inventors used PCR using degenerate PPO primers to screen apple fornovel PPO gene sequences. Using this approach, GPO3, APO5 and AP14 wereidentified in genomic DNA; GPO3, APO5 and PPO2 (PPOJ) were identified inapple fruit and apple leaf cDNA; and GPO3 and pSR7 were identified in animmature apple fruit cDNA library (Eugentech).

TABLE I Genbank PPO Gene Sequence Source Accession Sequence APO5 Boss etal. 1995 L29450 Complete HR_PPO2 PPO3 Haruta et al. 1998 D87669 CompletePPO7 Haruta et al. 1998 D87670 PPO2 Kim et al. 2001 AF380300 CompleteHR_PPO3 PPOJ Okanagan Specialty Fruits pSR8 Robinson (WO9302195) A27663Partial GPO3 Boss (personal Partial communication) Complete HR_PPO1 AP14Okanagan Specialty Fruits pSR7 Robinson (WO9302195) A27661 PartialHR_PPO4/5, HR_PPO8 Complete Okanagan Specialty Fruits APO3 (5′ APO3 Boss(personal Partial sequence.) communication) APO9 (5′ APO9 Boss (personalPartial sequence.) communication) APO3 (3′ APO3 Boss (personal Partialsequence.) communication) APO9 (3′ APO3 Boss (personal Partialsequence.) communication)

The apple PPO gene sequences thus obtained (FIG. 2) were aligned usingClustal W™ (FIG. 3) and were sorted into four groups usingContigExpress™ (Vector NTI™ Suite 9.0.0, Invitrogen) and the groups werenamed for the PPO sequence type.

Referring to FIG. 3, sequences AP095 and AP093 are 5′ and 3′non-overlapping fragments of the same clone. Similarly, sequences AP035and AP033 are 5′ and 3′ non-overlapping fragments of the same clone. The3′ sequences are identical while the 5′ ends do not overlap. Based onthe 3′ sequence, it is assumed that the APO9 and APO3 clones are thesame. Where APO9 overlaps the GPO3 sequence, they are 87% identical.Where APO3 overlaps the GPO3 sequence, they are 94% identical.

According to the alignment results obtained, it is expected that certainsequences are likely the same gene, or are likely equivalent from anantisense point of view (see Table II).

TABLE II Antisense Group Targets PPO2 PPO2, PPOJ, pSR8 GPO3 GPO3, AP14,APO9, APO3 APO5 APO5, PPO3, PPO7 pSR7 pSR7

Accordingly, the four apple PPO gene groups are: APO5 (includes APO5,PPO3 and PPO7), GPO3 (includes GALPO3, APO3, APO9 and AP14), PPO2(includes PPO2 and pSR8) and pSR7 (FIG. 4). Alignment of the four PPOgenes (PPO2, GPO3, APO5 and pSR7) with Clustal W showed an overallhomology/identity between these sequences of 61 to 75% (see Table III).

TABLE III Homology/identity of the four apple PPO genes PPO2 GPO3 APO5pSR7 PPO2 100 61 63 66 GPO3 100 75 62 APO5 100 65 pSR7 100RNA Extraction from Tissue

Total RNA for RT-PCR was isolated from various apple tissues using anovel cellulose method (Weirsma 2001, submitted). Briefly, 2 g of frozenground tissue was weighed out into a frozen 50 ml centrifuge tube.Extraction buffer (10 ml) preheated to 65° C. was added and the mixturewas incubated for 1 minute at 65° C. to melt the tissue. The mixture wasextracted one time with 5 ml of chloroform:isoamyl alcohol (24:1) withshaking for 10 minutes at room temperature. After centrifugation, theaqueous layer was filtered through miracloth into a fresh 50 mlcentrifuge tube. Ethanol was added to the aqueous layer to a finalconcentration of 30% and 0.5 g CC41 (Whatman) cellulose powder wasadded. The mixture was shaken for 45 minutes on ice. The mixture wasapplied to a BioRad Econo-column and washed with approximately 250 ml ofSTE (30% STE: 0.1 M NaCl, 0.05 M Tris and 30% ethanol). After washing,the column was allowed to go dry and residual STE was purged from thecolumn using a 60 ml syringe. ssRNA was eluted from the column withthree elutions of 2, 1, and 1 ml of sterile double distilled water usingan air purge in between each elution. Eluates were collected into a cold50 ml centrifuge tube. Nucleic acids were precipitated by addition of1/10 volume of 3 M NaAc pH 5.2 and 2.5 volumes of 95% ethanol overnightat −20° C. After centrifugation, the pellet was washed with 70% ethanoland aspirated dry. The RNA was solubilized in 600 μl of sterileRNAse-free water and transferred to a microfuge tube.

The 50 ml centrifuge tube was rinsed with an additional 300 μl of waterwhich was also transferred to the microfuge tube (total volume inmicrofuge tube=900 μl). The ssRNA was selectively precipitated byaddition of ⅓ volume of 8 M LiCl (2M LiCl final) overnight at −20° C.After centrifugation to collect the ssRNA, the ssRNA was solubilized in400 μl of water. RNA was re-precipitated by addition of 40 μl of 3 MNaAc pH 5.2 and 1 ml of 95% ethanol. The RNA pellet was washed with 70%ethanol, aspirated dry and solubilized in 50 μl of water. The RNA wasquantified spectrophotometrically and a sample was run on 1% TAE agarosegel to check integrity of RNA. RNA was stored at −80° C. until used.

EB: 0.2 M glycine, 0.1 M Na₂HPO₄, 0.6 M NaCl, 2% SDS, 2% PVP-40 and 5%BME

Degenerate PPO-Specific PCR Primers

PPO-specific degenerate PCR primers were developed using CODEHOP.Briefly, a consensus PPO amino acid sequence was generated from analignment of the known apple PPO sequences (L29450, D87669, D87670,GALPO3) plus the sequences for apricot (AF020786), sweet potato(AB038994), pokeweed (D45385), tobacco (Y12501), tomato (Z12838), potato(U22922) and grape (Z27411). The alignment was submitted to the BLOCKSmultiple alignment processor for arrangement into a format that isaccepted by CODEHOP. The BLOCKS output was submitted to CODEHOP forselection of degenerate primers, using the Malus domestica codon tablefor back translation. CODEHOP degenerate primers were selected that werewithin the Copper binding sites and had similar melting temperatures(Tms). Primer JCA1 (5′ TCT TCT TCC CNT TCC ACC GTT ACt ayy tnt ayt t 3′)[SEQ ID NO: 69] and primer JCB1 (5′ CCA GCG GAG TAA AAA TTC ccc atr tcytc 3′) [SEQ ID NO: 70] were selected. The primer JCA1 was modified toreflect codon usage in apple using the known apple DNA sequences.

Reverse Transcription

Reverse transcription (RT) (first strand synthesis) was carried outusing Superscript™ II reverse transcriptase according to themanufacturers' instructions (Invitrogen). Briefly, an RNA/primer mixturewas made that contained: 1 μg of total RNA, 1 μl of 10 mM dNTP and 1 μlof 2 μM cDNA primer in a final volume of 10 μl. The mixture was incubateat 65° C. for 5 minutes and then placed on ice for at least 1 minute. Tothis, 9 μl of a first strand synthesis reaction mixture containing: 4 μlof 5×RT buffer, 2 μl of 50 mM MgCl₂, 2 μl of 0.1 M DTT and 1 μl of RNAseOut™ was added. The RT reaction mixture was mixed gently, collected bybrief centrifugation and incubated at 42° C. for 2 minutes. To thereaction, 1 μl of Superscript II reverse transcriptase was added and thereaction was incubated at 42° C. for 50 minutes. The reaction wasterminated at 70° C. for 15 minutes and cooled on ice. Finally, 1 μl ofRNAse H was added and the reaction was incubated at 37° C. for 20minutes. The cDNA was used directly for PCR.

Hot Start, Touchdown PCR, TA Cloning and Sequencing

Hot start, touchdown PCR was used to amplify chromosomal DNA and cDNAsamples. For amplification of genomic DNA, the PCR reaction contained:1×PCR buffer, 1.5 mM MgCl₂, 200 μM dNTP, 1 μM JCA1, 1 μM JCB1, 1.25 UAmpliTaq Gold™, and 100 ng Golden Delicious genomic DNA. Foramplification of cDNA, the PCR reaction contained: lx PCR buffer, 1.5 mMMgCl₂, 200 μM dNTP, 1 μM PPO upper, 1 μM PPO lower, 1.25 U AmpliTaqGold, and 2 μl of cDNA. The reaction was overlaid with oil and cycled.After an initial hot start incubation for 9 minutes at 95° C., the PCRreaction was subjected to 1 cycle of 95° C., 1 min; 70° C., 1 min; and72° C., 1 min. The initial annealing temperature was reduced in eachsuccessive cycle by 2° C. to 62° C. The PCR reaction was subjected to atotal of 40 cycles. After cycling, the PCR reaction was subjected to afinal extension for 10 minute at 72° C., and then held at 6° C.

PCR products were size fractionated on TAE-agarose. Amplificationproducts were excised from the gel, gel cleaned and ligated into pGEM-TEasy™. The ligation reaction was electroporated into Electromax™ DH10bcells. Plasmids carrying inserted were isolated and the insert wassequenced using M13F and M13R primers (BigDye™, ABI).

3. Reduction of PPO Expression in Apples

In a pivotal review RNA interference, Sharp (2001) suggested that for atransgene to induce silencing of a related but not identical targetgene, the two segments must share regions of “identical anduninterrupted sequences of significant length” in the order of 30-35base pair at a minimum.

Since dsRNA is processed to 21-23 nucleotide segments, Sharp suggeststhat a single basepair mismatch between the siRNA and target RNAdramatically reduce gene targeting and silencing.

The inventors compared pair-wise, the four PPO apple sequences, within asliding (conservative) 22 base-pair window for regions of 100% homology.

This pair-wise analysis demonstrated that GPO3 and APO5, having anoverall sequence similarity of 75%, have several regions of identicaland uninterrupted sequences of significant length as shown below.

TABLE IIIb Number of Regions of 22 bp Micro Homology GPO3 APO5 pSR7 PPO20 0 0 GPO3 25 1 APO5 0Table IIIb shows that: there are no regions of 100% micro homologybetween PPO2 and GPO3, APO5 or pSR7; there are no regions of 100% microhomology between APO5 and pSR7; and that there are 25 regions of 100%micro homology of 22 bp between GPO3 and APO5. These are part of 3larger regions of 100% homology. There is 1 region of 100% microhomology of 22 bp between GPO3 and pSR7.4. Construction of PPO Suppression Transgene

In an illustrative example, the inventors used a PPO suppressiontransgene PGAS (PPO2, GPO3, APOS and pSR7), cloned in the senseorientation in the pBINPLUS to create GEN-03, to suppress PPO mRNAexpression of all four PPO isoenzymes.

The PGAS suppression transgene was constructed using standard molecularbiology techniques (Sambrook et al. 1989). Briefly, approximately 0.45kb individual PPO fragments (PPOJ, GPO3, APO5 and pSR7) (FIG. 5A) wereamplified from genomic DNA (GPO3, APOS) or cDNA (PPOJ, pSR7) usingdegenerate PCR primers, where the fragments include the Copper A andCopper B binding sites. PPOJ and PPO2 are 90% identical and could be thesame gene, if for some reason the sequencing was poor. A person skilledin the art would expect that, from a functional perspective, suppressionof either gene (PPOJ or PPO2) would be reasonably expected to inducesuppression of the other. The fragments were cloned individually andthen combined into a single chimeric PPO suppression fragment (PGAS)(FIG. 6A). Since the PPO fragments used in the PGAS transgene wereinitially amplified using degenerate PCR primers, the 5′ and 3′ ends ofthe PPO fragments used in the PGAS transgene may not exactly match thesequence of the endogenous PPO gene (FIG. 7A to 7L).

These PPO gene fragments were in the “sense” orientation under thecontrol of the double enhanced CAMV35S promoter (P_(CAMV35S)) andNopaline Synthase terminator (T_(NOS)) to create the PPO suppressiontransgene (P_(CAMV35S):PGAS:T_(NOS)). Approximately 50 bp of the PPOJsequence was lost during the construction of the vector.

The P_(CAMV35S):PGAS:T_(NOS) transgene was transferred into pBINPLUS tocreate the plant transformation vector GEN-03 (FIG. 8A). GEN-03 wastransferred into Agrobacterium tumefaciens LBA4404 in preparation forplant transformation. The elements of the GEN-03 T-DNA region that aretransferred to the plant are described in (FIG. 9A-9C).

In another illustrative example, the inventors constructed an alternatePPO suppression transgene (PGAS2) (FIGS. 6B and 8B) having a 1771 bpchimeric PPO Suppression sequence comprising 200 bp fragment of each offour apple PPO genes (PPO2, GPO3, APOS and pSR7; FIG. 5B), followed atits 3′ terminal by an apple intron, followed at the intron's 3′ terminalby inverted 200 bp fragment of each of pSR7, APOS, GPO3 and PPO2 (thesame fragments as those used in 5′ of the intron). The fragments ofPPO2, GPO3, APO5 and pSR7 used in the PGAS2 transgene were aligned withtheir respective genes (FIG. 7M-7X). The elements of the PGAS2 transgeneare described in (FIG. 9D-9F); which was used to create the OSF-02transformation vector (FIG. 8B). RNA transcribed from this 1771 bpchimeric PPO Suppression sequence is expected to generate a dsRNA stemof 800 bp with an intron loop. The transgene was under the control ofthe BUL409s promoter and the Ubiquitin 3 terminator (Garbarino andBelknap 1994).

The BUL409s promoter is a polyubiquitin promoter from the wild potatoSolanum bulbocastanum containing 5′ regulatory sequences (784 bp), the5′ untranslated region (59 bp), an intron (535 bp) and an ubiquitincoding domain (228 bp) (Bill Belknap, USDA Albany).

The Ubiquitin 3 terminator is the polyubiquitin terminator from potatoSolanum tuberosum (DQ320121) (Garbarino and Belknap 1994).

5. Apple Transformation

A. Gen-03

The GEN-03 vector (having the P_(CAMV35S):PGAS:T_(NOS) transgene) wastransformed into apple varieties Golden Delicious (also abbreviated as“GD”), Granny Smith (also abbreviated as “GS”), Fuji (also abbreviatedas “Fu”) and Gala (also abbreviated as “Ga”), using Agrobacteriumtumefaciens LBA4404 transformation.

Briefly, leaves of 3-week-old apple tissue culture plantlets wereexcised and cut into segments perpendicular to the mid-rib. Leafsegments were inoculated with Agrobacterium tumefaciens LBA4404 carryingthe recombinant vector GEN-03 at a density of 3×10⁸ cells/ml for 5 to 10minutes. Leaf segments were blotted on filter paper to remove excessbacterial cells and placed onto co-cultivation medium with the adaxialsurfaces in contact with the medium for 4 days (dark). Infected leafsegments were washed and placed onto regeneration medium containing 6mg/ml kanamycin with the adaxial surfaces in contact for 4 weeks (2weeks dark, 2 weeks light). Leaf segments were transferred toregeneration medium containing 50 mg/ml kanamycin (4 weeks). Transformedshoots were transferred to proliferation medium with 50 mg/ml kanamycin(4 weeks). Surviving shoots were transferred to proliferation medium.

The selection marker (NptII) confers resistance to the antibiotickanamycin. Cells that received and integrated the selection markerobtained the ability to regenerate in the presence of kanamycin. Shootsthat arose from callus after the transformation process typically arosefrom a single cell that integrated the selection marker. These shootswere presumed to be homogenous.

Each shoot represented a unique transformation event and was geneticallydistinct. Individual shoots were given unique EventID numbers toidentify the distinct genetic event (see Table IV). All plant material(tissue culture plants, field trees, tissue samples and apples) thatarose from these single genetics events retained the EventID number.

TABLE IV EventID and PlantID Numbers (GEN-03) Event A geneticallydistinct transformation event. EventID Event Identification Number. Aunique identification number assigned to each Event. Example: 705 PlantA tree either self-rooted or grafted. PlantID Plant IdentificationNumber. A unique identification number assigned to each Plant. Example:705-0001 The first plant of event number 705B. OSF-02

The OSF-02 vector (having the PBUL409s:PGAS2:TUBI3 transgene) wastransformed into apple varieties Golden Delicious, Granny Smith, Fujiand Gala, using Agrobacterium tumefaciens LBA4404 transformation.

Briefly, leaves of specially prepared Leaf Expansion Culture plants wereexcised and wounded with non-traumatic forceps (manufacturer). Woundedleaves were inoculated with Agrobacterium tumefaciens LBA4404 carryingthe recombinant vector OSF-02 for 5 minutes. Leaves are blotted onfilter paper to remove excess bacterial cells and placed ontoco-cultivation medium right side up for 3 days at 25° C. (dark).Infected leaves were washed. The tip and base were cut from each leafand the remaining leaf sliced into three sections. Leaf segments weretransferred to regeneration medium (without antibiotics or otherselection agents) and set in the dark for 3 weeks. Plates containing thetransformed leaf segments were transferred to the growth room (withoutlight). After 1 week, the lights were turned on. Over the next 3 to 6weeks, regenerating shoots were transferred to proliferation medium(Cornell University).

Genetically modified plants were identified by PCR.

Sterile techniques may be used as described herein.

Alternatively, using sterile techniques in a laminar flow hood, 3-4fully expanded, young leaves (10-20 mg) in good condition were takenfrom shoot cultures that were 3-4 weeks old and placed into labelled96-Deepwell plates. The plates were transferred to the −80° C. freezerand then freeze dried for 24 hours at <100 m Torr. Plant genomic DNA wasisolated from leaf tissue on an automated DNA extraction system usingthe “Slipstream MES” protocol (HortResearch).

Genomic DNA was amplified in a PCR reaction using PCR primers that arespecific for endogenous APO5, the PGAS or PGAS2 transgene, the nptIIselection marker (GEN-03) or vector backbone (OSF-02) (TABLE V-b1 andV-b2).

The BUL409s promoter (OBI-04/1313-1706) is unique to the PGAS2transgene.

The backbone nptII sequence (nptII Backbone set 1) is present in thebackbone of the OSF-02 vector. This sequence is not normally present inapple and will only be present in transgenic Events where the LB wasbypassed (subjected to read through) during T-DNA transfer (duringtransformation).

6. PCR Screening for Genetically Modified Lines

Genetically modified lines were identified by PCR. Briefly, usingsterile techniques in the laminar flow hood, 3-4 fully expanded, youngtissue culture leaves in good condition were taken from shoot culturesthat were 3-4 weeks old and placed into labeled FastPrep™ tubes. Eachtube was placed immediately into liquid nitrogen and each batch of tubeswas then transferred to the −80° C. freezer for storage. Plant genomicDNA for PCR was isolated from leaf tissue using CTAB (Lodhi al. 1994).Genomic DNA was amplified in a PCR reaction using the following PCRprimers that are specific for endogenous APO5, the PGAS transgene or thenptII selection marker of GEN-03 (TABLE V-al and V-a2). For PCR primersused to screen lines for OSF-02, see (TABLE V-b1 and V-b2).

TABLE V Table V-a1: PCR primers for GEN-03 Size Genotype Target Primers(bp) Screen for Presence of Correct APO5 Apo5 Forward/ 250PCR Positive Control Positive (Set1) Apo5 Reverse APO5 JA4/JA5 800PCR Positive Control Positive (Set2) PGAS CAMV35s/GPO3-R 953CAMV35S:PPO2 Junction Positive (Set1) PGAS GPO3-L/APO5-R 672GPO3:APO5 Junction Positive (Set2) PGAS pSR7-F (A81)/ 556pSR7:NOSTERM Junction Positive (Set3) NOSTERM nptII nptII Forward/ 286nptII Selection Marker Positive (set1) nptII Reverse nptIInptII-F/nptII-R 483 nptII Selection Marker Positive (Set2)Table V-a2: Primer Sequences (GEN-03) SEQ ID Target Primer Name NO:Primer Sequence (5′ to 3′) APO5 (Set1) Apo5 Forward 71GCGTTGATTGTGGTTTCCTT Apo5 Reverse 72 TCCCGTTCCACCGTTACTAC APO5 (Set2)JA4 73 GCC GTC GAC CGA CGA CGA CCC ACG JA5 74GCC GTC GAC AGC TGA GCC CAA GGA ATG PGAS (Set1) CAMV35s 75ACA ATC CCA CTA TCC TTC GC GPO3-R 76 CCT GGA TCT GGT TCA GTG CPGAS (Set2) GPO3-L 77 TTC GCT AAC CCG GAC TCT APO5-R 78CGG GTT CCC AAA GAA CAA CTT A PGAS (Set3) pSR7-F (A81) 79GCC AAG CTT TTC CTT TCC ACC GCA TGT NOSTERM 80TAT GAT AAT CAT CGC AAG AC nptII nptII Forward 81CCT GCT TGC CGA ATA TCA T (Set 1) nptII Reverse 82GAA ATC TCG TGA TGG CAG GT nptII nptII-F 83GAA CAA GAT GGA TTG CAC GCA G (Set 2) nptII-F 84CTG ATG CTC TTC GTC CAG ATC A Table V-b1: PCR primers for OSF-02 ProductGenotype Target Primers Size (bp) Screen for Presence of Correct APO5Apo5 Forward/ 250 PCR Positive Control Positive Apo5 Reverse BUL409sSP/OBI-04/1313-1706/ 394 PGAS2 Transgene Positive ASP/OBI-04/1313-1707nptII (Set 1) Left/Right 172 Vector Backbone Negative nptII(Set 2) Left/Right 228 Vector Backbone NegativeTable V-b2: Primer Sequences (OSF-02) SEQ ID Target Primer Name NO:Primer Sequence (5' to 3') APO5 Apo5 Forward 85GCG TTG ATT GTG GTT TCC TT Apo5 Reverse 86 TCC CGT TCC ACC GTT ACT ACBUL409s SP/OBI-04/1313-1706 87 AGG GAG TGT GAA AAG CCC TAASP/OBI-04/1313-1707 88 GGG GAG TTT GAA GTC GAT GA nptII Left 89GAA AGC TGC CTG TTC CAA AG (Set 1) Right 90 GAA AGA GCC TGA TGC ACT CCnptII Left 91 CGG CTC CGT CGA TAC TAT GT (Set 2) Right 92GCA GCG GTA TTT TTC GAT CA

APO5 is normally present in the genomic DNA of apple. A controlamplification of an 800 bp fragment from apple genomic DNA withAPOS-specific primers (JA4/JA5) showed that the particular DNA samplewas amplifiable and that all PCR reaction components were in workingorder. Accordingly, all apple genomic DNA samples were expected to yieldan 800 bp PCR fragment when amplified with these primers and onlygenomic DNA samples from which the APOS was amplified were analyzedfurther.

While APO5 is present in the genomic DNA of untransformed plants, theCAMV35s:PPO2 junction, (CAMV35s/GPO3-R), the GPO3:APO5 junction(GPO3-L/APO5-R), and the pSR7:NOSTERM junction (pSR7-F/NOSTERM) areunique to the PGAS Transgene.

nptII is not normally present in untransformed apple tissue.Amplification of a 483 bp fragment from apple genomic DNA withNptII-specific primers (NptII-f/NptII-R) was evidence that NptII waspresent and that the tissue was therefore genetically modified tissue.

7. PPO Activity and Gene Expression

Briefly, using sterile techniques in the laminar flow hood, 6-10 fullyexpanded, young tissue culture leaves (approximately 10 mg) in goodcondition were taken from shoot cultures that were 3-4 weeks old andplaced into labeled FastPrep™ tubes. Each tube was placed immediatelyinto liquid nitrogen and each batch of tubes was then transferred to the−80° C. freezer for storage. Crude PPO was extracted from frozen groundleaf tissue. Total soluble protein was measured using BCA. Total PPOActivity was measured using 4-methyl catechol as substrate. Theprocedure was a modification of the Polyphenol Oxidase activity assay ofBroothaerts et al (2000) adapted to a microtitre plate format.

In tissue culture, a survey of 34 untransformed Golden Delicious controlsamples, taken throughout the year, but from young healthy culture grownin nearly identical conditions, gave a PPO Specific Activity average of2613+/−1019, with Specific Activity values ranging from 774-5995 U/mgprotein. The Experimental Error of the PPO Assay was approximately5-10%. Events were subjected to PPO activity screening preferably twotimes and more preferably >two times at successive sub-culture points.

Total RNA was extracted using a small-scale modification of thecellulose-binding method of Fils-Lycaon et al. (1996). Two g of powderedapple tissue in a 50 ml polypropylene Oak Ridge tube were shaken at roomtemperature for 45 min with: 9.33 ml GPS buffer (0.2 M Glycine, 0.1 Msodium phosphate (dibasic), 0.6 M NaCl, pH 9.5); 1 ml 20% (w/v) SDS; 0.3g polyvinylpyrrolidone (PVP-40); 0.75 ml 2-mercaptoethanol; and 4.5 mlbuffer-saturated (pH 8) phenol. Two ml of chloroform:isoamyl alcohol(24:1) were added and mixed briefly before centrifuging for 20 min at 14k×g and 2° C. in a JA17 rotor (Beckman). Ten ml of the aqueous upperlayer were carefully removed and filtered through Miracloth™ and 95%ethanol was added to bring the final ethanol concentration to 30% (4.5ml 95% ethanol added to 10 ml sample). A 0.5 g quantity of cellulose(Whatman CC 51) was added and the slurry was shaken for 45 min on ice tobind the RNA. The cellulose was pelleted by centrifugation for 2 min atroom temperature at 800×g, the supernatant discarded and the pelletresuspended in 40 ml 30% STE (30% (v/v) ethanol, 0.1 M NaCl, 50 mMTris-HCl (pH 8.0), 1 mM EDTA). This wash was repeated five times withthe final pellet resuspended in 25 ml 30% STE and poured into a sterile1.5×15 cm nylon fritted column. The cellulose in the column was washedwith an additional 200 ml of 30% STE and the residual buffer wasexpelled with air. Total RNA was eluted with RNase-free water, thenprecipitated with ethanol/sodium acetate in the cold and washed with 70%ethanol. The RNA was resuspended in water, residual cellulose wasremoved by centrifugation and the RNA quantified with RiboGreen™(Invitrogen, Carlsbad, Calif.). cDNA was synthesized following a DNase Idigestion from 1 μg of total RNA using Superscript II (Invitrogen) andoligo(dT) by a modification of the protocol of Huang et al. (2000). EDTAwas added to chelate Mg before denaturation of the DNase I andadditional Mg was added with the RT buffer to bring the final unchelatedconcentration to 5 mM. The RNase H step was omitted. The cDNA wasdiluted 4-fold with water, EDTA added to give a slight excess (0.5 mM)over Mg and stored at −20 C until use.

Real time PCR analysis was conducted in: 1× AmpliTaq™ Gold Buffer II;2.5 mM MgCl2; 200 μM each dNTP; 7.5% glycerol; 3.0% DMSO; 1/40,000SYBRGreen II; 0.5 unit AmpliTaq Gold; 200 nM each primer; cDNAequivalent to 6.25 ng of starting total RNA; and 20 μl final volume.

Primers were designed using Primer 3 (Rozen and Skaletsky, 2000).Primers are given in Table VI.

TABLE VI PCR Primers (5′ to 3′) Size Target Primer One Primer Two (bp)PPO2 MaldoPPO2-69 [SEQ ID NO: 93] MaldoPPO2-71 [SEQ ID NO: 94] 177GGGACTCGCTCGACACTAAA TCACCTCGACGCTGATTGTA PPO2MaldoPPO2-69 [SEQ ID NO: 95] MaldoPPO2-70 [SEQ ID NO: 96] 229GGGACTCGCTCGACACTAAA TCGTCATGTGCCTTCTTCTG GPO3MaldoGPO3-64 [SEQ ID NO: 97] MaldoGPO3-65 [SEQ ID NO: 98] 163GTGAATGACGTGGACGATGA CATCATCTTCAGCACCCAAA GPO3MaldoGPO3-66 [SEQ ID NO: 99] MaldoGPO3-67 [SEQ ID NO: 100] 159CATCTTCAGCACCCAAATCC TGAATGACGTGGACGATGAG APO5MaldoAPO5-60 [SEQ ID NO: 101] MaldoAPO5-61 [SEQ ID NO: 102] 218AGTTTGCCGGAAGCTTTGTA TGATGCCTGGGTTGACATAA APO5MaldoAPO5-60 [SEQ ID NO: 103] MaldoAPO5-62 [SEQ ID NO: 104] 219AGTTTGCCGGAAGCTTTGTA TTGATGCCTGGGTTGACATA pSR7MaldopSR7-53 [SEQ ID NO: 105] MaldopSR7-54 [SEQ ID NO: 106] 183TAGTGTTCCGTGGCTGTTCA TCCTCCTCGTCGATCTTCTC pSR7MaldopSR7-53 [SEQ ID NO: 107] MaldopSR7-56 [SEQ ID NO: 108] 270TAGTGTTCCGTGGCTGTTCA CTGAGCGACTCAGCATCATC

A Stratagene Mx3000P instrument was used with cycle conditions of: 10min 95° C. initial denaturation/enzyme activation; 40 cycles of 30 s at95° C., 45 s at 60° C., 30 s at 72° C.; and with detection at the end ofthe 60° C. step. Dissociation curves were routinely run to ensure thatsingle products were produced. Baselines and thresholds were setmanually. Relative expression was calculated using the method of Pfaffl(2001) with efficiencies determined by the slopes of calibration curvesusing dilutions of cDNA as template. Normalization was done using anaverage of the expression values for the genes for protein disulphideisomerase (MdPDI1) and polyubiquitin (MdUBI2) which showed low variationin expression among the fruit samples. Measurement variation wasmaximized by using two cDNA synthesis reactions for each tissue andusing these in separate real time PCR runs for the duplicate valuesplotted. Relative copy numbers were plotted on a logarithmic scale.

8. Micrografting

Lines showing highly reduced total PPO activity in tissue culture weregrafted onto M9 (Mailing 9) rootstocks and advanced into field trialsaccording to Lane et al. (2003).

9. Controlled Bruising of Apples

Mature fruits were harvested from control and genetically modified applelines and returned to laboratory for analysis. Fruits were subjected toa series of tests to determine whether the expected reduced browningphenotype followed the marked reduction in total PPO gene expression andtotal PPO activity. The inventors measured gene expression byquantitative PCR, total PPO activity, and browning response to slicing,impact bruising and juicing.

A special bruising apparatus was designed at PARC Summerland to delivera controlled bruise to the apple with minimal destruction to the tissue.Apples were bruised in a consistent manner using the improvised ImpactDevice. Bruise response was reported as Change in Luminosity (ΔL) orTotal Change in Color (ΔE) between the bruised and non-bruised tissue asmeasured using a Minolta Chroma Meter.

10. Results

A total of 184 Events were determined to be genetically modified by PCRscreening. Of these, a total of 175 kanamycin-resistant Events weresubjected to PPO activity screening (Events 717, 720, 721, 723-727,735-737, 850, 881, 883, 884, 887 and 888 were not tested).

In one experiment, twelve GEN-03 Events plus untransformed controlGolden Delicious or Granny Smith were selected for field trials (FIG.10A). Some of these showed reduced-browning potential and others weresent as controls. In another experiment, twenty additional GEN-03 Eventswere selected for field trials (FIG. 10B). In other experiments, 10OSF-02 Events were selected for field trials (FIG. 10C). In anotherexperiment, 18 OSF-2 Events were selected for field trials (FIG. 10D).

Thirty-two GEN-03 Events were micrografted onto rootstocks (Malling 9),grown in the greenhouse/screenhouse and transferred into the field.Plants were grown in the field under standard commercial tree fruitmanagement conditions.

Control and genetically modified fruits were harvested from the fieldtrial and assessed. It is known that PPO gene expression is the highestand PPO protein is produced in immature fruit. Therefore, geneexpression and Total PPO activity were measured in immature fruitharvested in the spring.

Detailed data from eight Events is provided to illustrate therelationship between total PPO activity in tissue culture leaf material,gene expression and total PPO activity in immature fruit and the desirednon-browning phenotype achieved in mature apple fruit (FIG. 11).

Based on the herein described relationship between low tissue culturetotal PPO activity and the reduced-browning fruit phenotype, theinventors reasonably predicted that 13 Golden Delicious Events (702,703, 705, 707, 730, 743, 752, 792, 801, 831, 842, 845 and 846), 1 GrannySmith Event (784), and 1 Fuji Event (872) should produce areduced-browning fruit phenotype. In fact, Event 792 showed a reducedtotal PPO activity of 66% (FIG. 14B) and a ΔL of −1.1 and −0.5 in twoindependent experiments (FIG. 14C).

Three Golden Delicious Events (705, 707 and 743) had been selectedinitially showing reduction in total PPO activity in tissue culture ofapproximately 80%-90% relative to a control fruit. In immature fruittissue, these Events showed significant reduction in gene expression ofall four PPO genes. The suppression was more complete closer to the endsof the transgene and especially toward the 3′ end (pSR7). Decreased geneexpression in immature fruit tissue was reflected in marked reduction intotal PPO Activity of approximately 75-92%. Apples harvested from theseEvents were of a reduced-browning phenotype (low change in luminosity).Images of the “Controlled Bruising” provided in FIG. 12 clearly showthat the change in luminosity that was measured in the reduced-browningEvents was barely visible to the eye.

Juice produced from Event 743 did not significantly brown (FIG. 13 top).Even when left overnight at room temperature, while the untransformedcontrol was darkened considerably within 15 minutes. It was alsoobserved that the wet bruising often associated with damaged apple fleshdid not occur in the reduced-browning Events.

The results obtained for Golden Delicious apples were similarly obtainedin Granny Smith apple (784) (FIG. 13 bottom).

Many other Events were sent to the field for evaluation and the detailedresults are reported in FIGS. 14A-14C.

Recovery of a large number of Golden Delicious Events reflects theemphasis on this apple variety for proof of concept of thereduced-browning technology and the amount of Golden Delicious materialpushed through the transformation procedure, and should not be construedas limiting the invention.

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All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural reference unless the contextclearly dictates otherwise. Unless defined otherwise all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

The invention claimed is:
 1. A genetically modified apple plant, whereinsaid apple plant is genetically modified to comprise at least oneheterologous nucleic acid molecule comprising at least 20 contiguousnucleotides 100% identical to at least 20 contiguous nucleotides of afirst nucleotide sequence and at least 20 contiguous nucleotides 100%identical to at least 20 contiguous nucleotides of a second nucleotidesequence, wherein the first and second nucleotide sequences are selectedfrom two different nucleotide sequences selected from the groupconsisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ IDNO:
 33. 2. A genetically modified cell, seed, seedling, part, tissue,fruit or progeny of the genetically modified apple plant of claim
 1. 3.The genetically modified apple plant of claim 1, wherein said appleplant is genetically modified to comprise at least one heterologousnucleic acid molecule comprising at least 50 contiguous nucleotides 100%identical to at least 50 contiguous nucleotides of the first nucleotidesequence and at least 50 contiguous nucleotides 100% identical to atleast 50 contiguous nucleotides of the second nucleotide sequence. 4.The genetically modified apple plant of claim 3, wherein said appleplant is genetically modified to comprise at least one heterologousnucleic acid molecule comprising at least 100 contiguous nucleotides100% identical to at least 100 contiguous nucleotides of the firstnucleotide sequence and at least 100 contiguous nucleotides 100%identical to at least 100 contiguous nucleotides of the secondnucleotide sequence.
 5. A genetically modified apple plant, wherein saidapple plant is genetically modified to comprise at least oneheterologous nucleic acid molecule comprising at least a firstnucleotide sequence and a second nucleotide sequence, wherein the firstand second nucleotide sequences are different from each other and areselected from the group consisting of nucleotide sequences that are atleast 90% identical to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, andSEQ ID NO:
 33. 6. The genetically modified apple plant of claim 5,wherein said apple plant is genetically modified to comprise at leastone heterologous nucleic acid molecule comprising at least a firstnucleotide sequence and a second nucleotide sequence, wherein the firstand second nucleotide sequences are different from each other and areselected from the group consisting of nucleotide sequences that are atleast 95% identical to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, andSEQ ID NO:
 33. 7. The genetically modified apple plant of claim 6,wherein said apple plant is genetically modified to comprise at leastone heterologous nucleic acid molecule comprising at least the firstnucleotide sequence and the second nucleotide sequence, wherein thefirst and second nucleotide sequences are selected from the groupconsisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ IDNO:
 33. 8. A nucleic acid construct comprising at least 20 contiguousnucleotides 100% identical to at least 20 contiguous nucleotides of afirst nucleotide sequence and at least 20 contiguous nucleotides 100%identical to at least 20 contiguous nucleotides of a second nucleotidesequence, wherein the first and second nucleotide sequences are selectedfrom two different nucleotide sequences selected from the groupconsisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ IDNO:
 33. 9. The nucleic acid construct of claim 8, further comprising atleast 20 contiguous nucleotides 100% identical to at least 20 contiguousnucleotides of a third nucleotide sequence, wherein the third nucleotidesequence is selected from the group consisting of SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and wherein the thirdnucleotide sequence is different from the first and second nucleotidesequences.
 10. The nucleic acid construct of claim 9, comprising atleast 20 contiguous nucleotides 100% identical to at least 20 contiguousnucleotides of SEQ ID NO: 30; at least 20 contiguous nucleotides 100%identical to at least 20 contiguous nucleotides of SEQ ID NO: 31; atleast 20 contiguous nucleotides 100% identical to at least 20 contiguousnucleotides of SEQ ID NO: 32; and at least 20 contiguous nucleotides100% identical to at least 20 contiguous nucleotides of SEQ ID NO: 33.11. A nucleic acid construct comprising at least a first nucleotidesequence and a second nucleotide sequence, wherein the first and secondnucleotide sequences are selected from two different nucleotidesequences selected from the group consisting of sequences that are 90%identical to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO:33.
 12. The nucleic acid construct of claim 11, wherein the firstnucleotide sequence and the second nucleotide sequence are at least 95%identical to two different nucleotide sequences selected from the groupconsisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ IDNO:
 33. 13. The nucleic acid construct of claim 11, wherein the firstnucleotide sequence and the second nucleotide sequence are two differentnucleotide sequences selected from the group consisting of SEQ ID NO:30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.